JP7796264B2 - Pharmaceutical composition containing nitrogen-containing saturated heterocyclic derivative - Google Patents

Description

The present invention relates to a nitrogen-containing saturated heterocyclic derivative or a pharmaceutically acceptable salt thereof that inhibits or reduces the accumulation of abnormal protein aggregates in the brain, as well as a therapeutic and/or preventive agent for central nervous system disorders involving abnormal protein aggregates in the brain, which contains the derivative as an active ingredient. The present invention also provides a method for reproducing the pathology of Parkinson's disease using neural spheroids and a method for assessing the amount of α-synuclein aggregates using the same.

In neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis, abnormally aggregated proteins form in the brains of patients, and these aggregates are thought to exhibit neurotoxicity, causing the onset and progression of the disease.

The proteins that make up the aggregates vary depending on the disease, and α-synuclein has been reported to be the main component of the aggregates that cause Parkinson's disease. Abnormally aggregated α-synuclein is neurotoxic, and it has also been reported that aggregated α-synuclein propagates between nerve cells.

One treatment for Parkinson's disease is the administration of levodopa, a dopamine precursor, as a symptomatic treatment, but no fundamental cure has yet been established. In recent years, there has been vigorous research into the development of disease-modifying drugs for Parkinson's disease, but no drugs currently undergoing clinical trials have been reported that can effectively inhibit or reduce the accumulation of α-synuclein aggregates.

α-Synuclein aggregates are thought to be the underlying cause of Lewy body diseases, including Parkinson's disease (dementia with Lewy bodies, multiple system atrophy, Gaucher disease, infantile neuroaxonal dystrophy, etc.). Therefore, drugs that inhibit or reduce the accumulation of α-synuclein aggregates are expected to have an ameliorative effect on the pathology of these diseases.

To date, no in vitro evaluation system has been reported that reproduces the α-synuclein aggregates that occur endogenously within neurons. Evaluation systems for α-synuclein pathology have often been shown to measure the increase in phosphorylated α-synuclein levels upon addition of in vitro synthesized α-synuclein oligomers, making it impossible to evaluate the inhibitory effect on the accumulation of α-synuclein aggregates or the reduction of accumulated α-synuclein aggregates.

To date, drugs such as NPT200-11 (Neuropore) and Anle138b (MODAG) have been reported to inhibit α-synuclein aggregate formation, but these have been evaluated based on their ability to inhibit the aggregation of α-synuclein when it is artificially induced to aggregate in vitro (Patent Documents 1 and 2).

Furthermore, it has been reported that (4aR,8aS)-hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one derivatives, such as (4aR,8aS)-6-{4-[5-(trifluoromethyl)pyridin-3-yl]piperidine-1-carbonyl}hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one and (4aR,8aS)-6-[4-(5-ethylpyridin-3-yl)piperidine-1-carbonyl]hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one, have monoacylglycerol lipase (MAGL) inhibitory activity and are useful for neuroinflammation, neurodegenerative diseases, and the like (Patent Document 3). Furthermore, it has been reported that (azetidin-1-yl)(phenyl)methanone derivatives such as 2-chloro-3-{3-[6-(trifluoromethyl)pyridin-3-yl]azetidine-1-carbonyl}-4-[(1,1,1-trifluoropropan-2-yl)oxy]benzonitrile and 2-methoxy-3-{3-[6-(trifluoromethyl)pyridin-3-yl]azetidine-1-carbonyl}-4-[(1,1,1-trifluoropropan-2-yl)oxy]benzonitrile have glycine transporter 1 (GlyT1) inhibitory activity and are useful for treating neurodegenerative diseases and the like (Patent Document 4).
However, these compounds are all different from the nitrogen-containing saturated heterocyclic derivatives of the present invention. Furthermore, these documents do not disclose or suggest anything about the nitrogen-containing saturated heterocyclic derivatives of the present invention. Furthermore, they do not suggest anything about the inhibitory or reducing effect on the accumulation of abnormal protein aggregates in the brain.

International Publication No. 2011/084642 International Publication No. 2010/000372 International Publication No. 2019/180185 International Publication No. 2016/073420

The present invention aims to provide a compound or a pharmaceutically acceptable salt thereof, a composition containing said compound, etc., for use in the prevention or treatment of central nervous system disorders characterized by the inhibitory or reduction of the accumulation of abnormal protein aggregates in the brain. It also aims to provide a method for reproducing the pathology of Parkinson's disease using neural spheroids and a method for assessing the amount of α-synuclein aggregates using said method.

As a result of extensive research, the present inventors have discovered that a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof (hereinafter sometimes abbreviated as "the compound of the present invention" as necessary) has the effect of inhibiting or reducing the accumulation of abnormal protein aggregates in the brain, and have also discovered a method for reproducing the pathology of Parkinson's disease using neural spheroids and a method for assessing the amount of α-synuclein aggregates using the same, thereby completing the present invention. Specifically, the present invention is as follows:

[Section 1]
Formula (1):
[In the formula,
X represents oxygen or ^NR7 ;
R ⁷ represents hydrogen, C _1-3 alkyl optionally substituted with 1 to 3 halogen atoms which may be the same or different, or cyclopropyl;
Y represents CH or nitrogen;
m represents 0, 1 or 2;
n represents 0 or 1;
r represents 0, 1, 2, 3 or 4;
s represents 0, 1 or 2;
(wherein when s=0, Y is CH and r is 1, 2, 3, or 4;
when s=1, Y is CH and r is 0, 1, 2, or 3;
When s=2, r is 1 or 2.
R ¹ represents hydrogen, halogen, methyl or hydroxy;
^R2 represents hydrogen, halogen, methyl or hydroxy;
^R3 represents hydrogen or _C1-3 alkyl;
^R4 represents hydrogen or _C1-3 alkyl;
wherein ^R3 and ^R4 together may form a bridged methylene or ethylene;
^R5 represents halogen, _C1-3 alkyl optionally substituted with 1 to 3 of the same or different halogens, or _C1-3 alkoxy optionally substituted with 1 to 3 of the same or different halogens;
R ⁶ represents hydrogen, halogen, C _1-3 alkyl optionally substituted with 1 to 3 of the same or different halogens, or C _1-3 alkoxy optionally substituted with 1 to 3 of the same or different halogens;
Hy represents a pyridine ring, a pyridazine ring, a pyrimidine ring, or a pyrazine ring;
Here,
(I) When Hy including ^R5 and ^R6 is 5-fluoropyridin-2-yl and m and n are 1, r is 0 and s is 1;
(II) When Hy including ^R5 and ^R6 is 6-methoxypyridin-3-yl, m and n are 1, and X is oxygen, r is 0 and s is 1;
(III) When Hy including ^R5 and ^R6 is 5-methoxypyridin-2-yl, m and n are 1, and X is ^NR7 , then r is 0 and s is 1;
(IV) When ^R5 is methyl and ^R6 is hydrogen, r is 0 and s is 1;
however,
(I') Hy including ^R5 and ^R6 is not 4,6-dimethylpyrimidin-2-yl or 5-bromopyrimidin-2-yl;
(II') When Hy including ^R5 and ^R6 is 5-chloropyridin-2-yl, m and n are 1, and ^R1 and ^R2 are not both hydrogen;
However, the following compounds (III') (1-isopropylpiperidin-4-yl){3-(2-methoxypyridin-3-yl)pyrrolidin-1-yl}methanone and (IV') (3-ethoxyoxetan-3-yl)[3-{4-(trifluoromethyl)pyrimidin-2-yl}pyrrolidin-1-yl]methanone,
excluding
A pharmaceutical composition comprising a compound represented by the formula: or a pharmaceutically acceptable salt thereof.

[Section 2]
m is 1,
n is 1;
Item 1. The pharmaceutical composition according to Item 1.

[Section 3]
R ¹ and R ² are each independently hydrogen, methyl, or fluorine;
Item 3. The pharmaceutical composition according to Item 1 or 2.

[Section 4]
R ¹ and R ² are hydrogen;
Item 3. The pharmaceutical composition according to Item 1 or 2.

[Section 5]
X is oxygen, NH or NMe;
Item 5. The pharmaceutical composition according to any one of Items 1 to 4.

[Section 6]
^R3 is hydrogen;
Item 6. The pharmaceutical composition according to any one of Items 1 to 5.

[Section 7]
^R4 is methyl or ethyl;
Item 7. The pharmaceutical composition according to any one of Items 1 to 6.

[Section 8]
Y is CH;
Item 8. The pharmaceutical composition according to any one of Items 1 to 7.

[Section 9]
s is 1,
Item 9. The pharmaceutical composition according to any one of Items 1 to 8.

[Section 10]
r is 0 and s is 1;
Item 10. The pharmaceutical composition according to any one of Items 1 to 9.

[Section 11]
Formula (2):
[In the formula,
X represents oxygen, NH or NMe;
^R4 represents methyl or ethyl;
^R5 represents halogen, _C1-3 alkyl optionally substituted with 1 to 3 of the same or different halogens, or _C1-3 alkoxy optionally substituted with 1 to 3 of the same or different halogens;
R ⁶ represents hydrogen, halogen, C _1-3 alkyl optionally substituted with 1 to 3 of the same or different halogens, or C _1-3 alkoxy optionally substituted with 1 to 3 of the same or different halogens;
Hy represents a pyridine ring, a pyridazine ring, a pyrimidine ring, or a pyrazine ring;
however,
Hy including ^R5 and ^R6 is not 5-chloropyridin-2-yl.
Item 1. The pharmaceutical composition according to item 1,

[Section 12]
Hy is a pyridine ring;
Item 12. The pharmaceutical composition according to any one of Items 1 to 11.

[Section 13]
^R5 is trifluoromethyl;
Item 13. The pharmaceutical composition according to any one of Items 1 to 12.

[Section 14]
Hy is pyridin-3-yl;
Item 14. The pharmaceutical composition according to any one of Items 1 to 13.

[Section 15]
X is oxygen;
Item 15. The pharmaceutical composition according to any one of Items 1 to 14.

[Section 16]
^R4 is methyl;
Item 16. The pharmaceutical composition according to any one of Items 1 to 15.

[Section 17]
X is NH or NMe;
Item 15. The pharmaceutical composition according to any one of Items 1 to 14.

[Section 18]
X is NMe;
Item 15. The pharmaceutical composition according to any one of Items 1 to 14.

[Section 19]
Item 1, wherein the compound is selected from the following group of compounds:
(3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Example 1),
(3-methyloxetan-3-yl){4-[5-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Example 2),
(3-methyloxetan-3-yl){4-[4-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Example 3),
(3-methyloxetan-3-yl){4-[2-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Example 4),
{4-[5-fluoro-6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}(3-methyloxetan-3-yl)methanone (Example 29),
(3-methyloxetan-3-yl){4-[4-methyl-6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Example 31),
(1,3-dimethylazetidin-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Example 41),
(1,3-dimethylazetidin-3-yl){4-[2-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Example 42),
(1,3-Dimethylazetidin-3-yl){4-[4-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Example 43), and (1,3-Dimethylazetidin-3-yl){4-[5-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Example 44).

[Section 20]
Item 1, wherein the compound is selected from the following group of compounds:
(3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Example 1),
(3-methyloxetan-3-yl){4-[5-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Example 2),
{4-[5-fluoro-6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}(3-methyloxetan-3-yl)methanone (Example 29),
(3-methyloxetan-3-yl){4-[4-methyl-6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Example 31),
(1,3-Dimethylazetidin-3-yl){4-[2-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Example 42), and (1,3-Dimethylazetidin-3-yl){4-[5-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Example 44).

[Section 21]
Item 21. A therapeutic or preventive agent for a central nervous system disease associated with abnormal protein aggregates in the brain, comprising the pharmaceutical composition according to any one of Items 1 to 20.

[Section 22]
Item 22. The therapeutic or prophylactic agent according to Item 21, wherein the central nervous system disease associated with abnormal aggregates of proteins in the brain is a central nervous system disease associated with tau, α-synuclein, TDP-43, or polyglutamine.

[Section 23]
Item 22. The therapeutic or prophylactic agent according to Item 21, wherein the central nervous system disease associated with abnormal protein aggregates in the brain is Alzheimer's disease, frontotemporal lobar degeneration, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Gaucher disease, infantile neuroaxonal dystrophy, amyotrophic lateral sclerosis, Huntington's disease, or spinocerebellar ataxia.

[Section 24]
Item 22. The therapeutic or prophylactic agent according to Item 21, wherein the central nervous system disease associated with abnormal protein aggregates in the brain is a central nervous system disease associated with α-synuclein.

[Section 25]
Item 22. The therapeutic or prophylactic agent according to Item 21, wherein the central nervous system disease associated with abnormal intracerebral protein aggregates is Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Gaucher disease, or infantile neuroaxonal dystrophy.

[Section 26]
A method for treating or preventing a central nervous system disease associated with abnormal aggregation of proteins in the brain, comprising administering to a patient in need of treatment a therapeutically effective amount of the compound according to any one of items 1 to 20 or a pharmaceutically acceptable salt thereof.

[Section 27]
Item 21. Use of the pharmaceutical composition according to any one of Items 1 to 20 for the manufacture of a therapeutic or preventive agent for a central nervous system disease associated with abnormal protein aggregates in the brain.

[Section 28]
Item 21. The pharmaceutical composition according to any one of Items 1 to 20, for use in treating or preventing a central nervous system disease associated with abnormal protein aggregates in the brain.

[Section 29]
Item 21. A therapeutic or preventive agent for a central nervous system disease associated with abnormal aggregation of brain proteins, comprising the pharmaceutical composition according to any one of Items 1 to 20 in combination with at least one drug selected from the group consisting of L-dopa, a dopamine agonist, an MAO-B inhibitor, a catechol-O-methyltransferase (COMT) inhibitor, an αSyn antibody, and a pharmaceutically acceptable salt thereof.

[Section 30]
Item 22. The therapeutic or prophylactic agent according to Item 21, for treating or preventing a central nervous system disease associated with abnormal aggregation of brain proteins, in combination with at least one drug selected from the group consisting of L-dopa, a dopamine agonist, an MAO-B inhibitor, a catechol-O-methyltransferase (COMT) inhibitor, an αSyn antibody, and a pharmaceutically acceptable salt thereof.

[Section 31]
Formula (1):
[In the formula,
X represents oxygen or ^NR7 ;
R ⁷ represents hydrogen, C _1-3 alkyl optionally substituted with 1 to 3 halogen atoms which may be the same or different, or cyclopropyl;
Y represents CH or nitrogen;
m represents 0, 1 or 2;
n represents 0 or 1;
r represents 0, 1, 2, 3 or 4;
s represents 0, 1 or 2;
(wherein when s=0, Y is CH and r is 1, 2, 3, or 4;
when s=1, Y is CH and r is 0, 1, 2, or 3;
When s=2, r is 1 or 2.
R ¹ represents hydrogen, halogen, methyl or hydroxy;
^R2 represents hydrogen, halogen, methyl or hydroxy;
^R3 represents hydrogen or _C1-3 alkyl;
^R4 represents hydrogen or _C1-3 alkyl;
wherein ^R3 and ^R4 together may form a bridged methylene or ethylene;
^R5 represents hydrogen, halogen, _C1-3 alkyl optionally substituted with 1 to 3 of the same or different halogens, or _C1-3 alkoxy optionally substituted with 1 to 3 of the same or different halogens;
R ⁶ represents hydrogen, halogen, C _1-3 alkyl optionally substituted with 1 to 3 of the same or different halogens, or C _1-3 alkoxy optionally substituted with 1 to 3 of the same or different halogens;
Hy represents a pyridine ring, a pyridazine ring, a pyrimidine ring, or a pyrazine ring.
A therapeutic or preventive agent for a central nervous system disease associated with abnormal aggregation of proteins in the brain, comprising, as an active ingredient, a compound represented by the formula:

[Section 32]
m is 1,
n is 1;
Item 32. The therapeutic or prophylactic agent according to Item 31.

[Section 33]
R ¹ and R ² are each independently hydrogen, methyl, or fluorine;
Item 33. The therapeutic or prophylactic agent according to Item 31 or 32.

[Section 34]
R ¹ and R ² are hydrogen;
Item 33. The therapeutic or prophylactic agent according to Item 31 or 32.

[Section 35]
X is oxygen, NH or NMe;
Item 35. The therapeutic or prophylactic agent according to any one of Items 31 to 34.

[Section 36]
^R3 is hydrogen;
Item 36. The therapeutic or prophylactic agent according to any one of Items 31 to 35.

[Section 37]
^R4 is methyl or ethyl;
Item 37. The therapeutic or prophylactic agent according to any one of Items 31 to 36.

[Section 38]
Y is CH;
Item 38. The therapeutic or prophylactic agent according to any one of Items 31 to 37.

[Section 39]
s is 1,
Item 39. The therapeutic or prophylactic agent according to any one of Items 31 to 38.

[Section 40]
r is 0 and s is 1;
Item 40. The therapeutic or prophylactic agent according to any one of Items 31 to 39.

[Section 41]
Formula (2):
[In the formula,
X represents oxygen, NH or NMe;
^R4 represents methyl or ethyl;
^R5 represents hydrogen, halogen, _C1-3 alkyl optionally substituted with 1 to 3 of the same or different halogens, or _C1-3 alkoxy optionally substituted with 1 to 3 of the same or different halogens;
R ⁶ represents hydrogen, halogen, C _1-3 alkyl optionally substituted with 1 to 3 of the same or different halogens, or C _1-3 alkoxy optionally substituted with 1 to 3 of the same or different halogens;
Hy represents a pyridine ring, a pyridazine ring, a pyrimidine ring, or a pyrazine ring.
Item 32. The therapeutic or prophylactic agent according to Item 31, represented by the formula:

[Section 42]
Hy is a pyridine ring;
Item 42. The therapeutic or prophylactic agent according to any one of Items 31 to 41.

[Section 43]
^R5 is trifluoromethyl;
Item 43. The therapeutic or prophylactic agent according to any one of Items 31 to 42.

[Section 44]
Hy is pyridin-3-yl;
Item 44. The therapeutic or prophylactic agent according to any one of Items 31 to 43.

[Section 45]
X is oxygen;
Item 45. The therapeutic or prophylactic agent according to any one of Items 31 to 44.

[Section 46]
^R4 is methyl;
Item 46. The therapeutic or prophylactic agent according to any one of Items 31 to 45.

[Section 47]
X is NH or NMe;
Item 45. The therapeutic or prophylactic agent according to any one of Items 31 to 44.

[Section 48]
X is NMe;
Item 45. The therapeutic or prophylactic agent according to any one of Items 31 to 44.

[Section 49]
Item 49. The therapeutic or prophylactic agent according to any one of Items 31 to 48, wherein the central nervous system disease associated with abnormal aggregates of proteins in the brain is a central nervous system disease associated with tau, α-synuclein, TDP-43, or polyglutamine.

[Section 50]
Item 49. The therapeutic or prophylactic agent according to any one of Items 31 to 48, wherein the central nervous system disease associated with abnormal protein aggregates in the brain is Alzheimer's disease, frontotemporal lobar degeneration, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Gaucher disease, infantile neuroaxonal dystrophy, amyotrophic lateral sclerosis, Huntington's disease, or spinocerebellar ataxia.

[Section 51]
Item 49. The therapeutic or prophylactic agent according to any one of Items 31 to 48, wherein the central nervous system disease associated with abnormal protein aggregates in the brain is a central nervous system disease associated with α-synuclein.

[Section 52]
Item 49. The therapeutic or prophylactic agent according to any one of Items 31 to 48, wherein the central nervous system disease associated with abnormal protein aggregates in the brain is Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Gaucher disease, or infantile neuroaxonal dystrophy.

[Section 53]
A method for treating or preventing a central nervous system disease associated with abnormal aggregation of proteins in the brain, comprising administering to a patient in need of treatment a therapeutically effective amount of the compound according to any one of items 31 to 48 or a pharmaceutically acceptable salt thereof.

[Section 54]
Item 49. Use of the compound according to any one of Items 31 to 48 or a pharmaceutically acceptable salt thereof for the manufacture of a therapeutic or preventive agent for a central nervous system disease associated with abnormal intracerebral protein aggregates.

[Section 55]
Item 49. The compound or a pharmaceutically acceptable salt thereof according to any one of Items 31 to 48 for use in treating or preventing a central nervous system disease associated with abnormal protein aggregation in the brain.

[Section 56]
A therapeutic or preventive agent for a central nervous system disease associated with abnormal aggregation of brain proteins, comprising the therapeutic or preventive agent according to any one of Items 31 to 48 in combination with at least one drug selected from the group consisting of L-dopa, a dopamine agonist, an MAO-B inhibitor, a catechol-O-methyltransferase (COMT) inhibitor, an αSyn antibody, and a pharmaceutically acceptable salt thereof.

[Section 57]
Item 49. The therapeutic or prophylactic agent according to any one of Items 31 to 48, for treating or preventing a central nervous system disorder associated with abnormal aggregation of brain proteins, in combination with at least one drug selected from the group consisting of L-dopa, a dopamine agonist, an MAO-B inhibitor, a catechol-O-methyltransferase (COMT) inhibitor, an αSyn antibody, and a pharmaceutically acceptable salt thereof.

[Section 58]
A method for reproducing Parkinson's disease pathology by three-dimensionally culturing neural spheroids using synucleopathy-associated gene-mutated human iPS cells, comprising step (I);
(I) Measuring the amount of α-synuclein aggregates from neural spheroids.

[Section 59]
A method for evaluating a drug that inhibits or reduces the accumulation of α-synuclein aggregates in Parkinson's disease pathology by three-dimensionally culturing neural spheroids using human iPS cells with a mutated synucleopathy-related gene, comprising step (I);
(I) Measuring the amount of α-synuclein aggregates from neural spheroids.

[Section 60]
Formula (3):
[In the formula,
^R4 represents methyl or ethyl;
^R5 represents trifluoromethyl;
R ⁶ represents hydrogen, halogen, C _1-3 alkyl optionally substituted with 1 to 3 of the same or different halogens, or C _1-3 alkoxy optionally substituted with 1 to 3 of the same or different halogens;
Hy represents pyridin-3-yl.
or a pharmaceutically acceptable salt thereof,
A production method comprising the following step 1:
(Step 1) Formula (4):
[In the formula, R ⁵ , R ⁶ , and Hy represent the same groups as above.]
or a salt thereof with a compound represented by formula (5):
[wherein R ⁴ represents the same group as above,
A represents OH or halogen.
or a salt thereof to produce a compound represented by formula (3) or a pharmaceutically acceptable salt thereof.

[Section 61]
Formula (3):
[In the formula,
^R4 represents methyl or ethyl;
^R5 represents trifluoromethyl;
R ⁶ represents hydrogen, halogen, C _1-3 alkyl optionally substituted with 1 to 3 of the same or different halogens, or C _1-3 alkoxy optionally substituted with 1 to 3 of the same or different halogens;
Hy represents pyridin-3-yl.
or a pharmaceutically acceptable salt thereof,
A production method comprising the following step 1:
(Step 1) Formula (4):
[In the formula, R ⁵ , R ⁶ , and Hy represent the same groups as above.]
or a salt thereof with a compound represented by formula (5):
[wherein R ⁴ represents the same group as above,
A represents OH.
or a salt thereof in the presence of 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide and triethylamine to produce a compound represented by formula (3) or a pharmaceutically acceptable salt thereof.

[Section 62]
Formula (6):
[In the formula,
^R4 represents methyl;
^R5 represents trifluoromethyl;
R ⁶ represents hydrogen, halogen, C _1-3 alkyl optionally substituted with 1 to 3 of the same or different halogens, or C _1-3 alkoxy optionally substituted with 1 to 3 of the same or different halogens;
Hy represents pyridin-3-yl.
or a pharmaceutically acceptable salt thereof,
A production method comprising the following steps 1 to 3:
(Step 1) Formula (4):
[In the formula, R ⁵ , R ⁶ , and Hy represent the same groups as above.]
or a salt thereof with a compound represented by formula (7):
[wherein R ⁴ represents the same group as above,
A represents OH or halogen;
Pro represents an amino-protecting group.
or a salt thereof to form a compound represented by formula (8):
[In the formula, R ⁴ , R ⁵ , R ⁶ , Hy, and Pro represent the same groups as above.]
or a salt thereof;
(Step 2) The protecting group of the amino group of the compound represented by formula (8) or a salt thereof is deprotected to obtain a compound represented by formula (9):
[In the formula, R ⁴ , R ⁵ , R ⁶ , and Hy represent the same groups as above.]
or a salt thereof;
(Step 3) A step of reacting a compound represented by formula (9) or a salt thereof with formaldehyde or an equivalent thereof in the presence of a reducing agent to produce a compound represented by formula (6) or a pharmaceutically acceptable salt thereof.

[Section 63]
Formula (6):
[In the formula,
^R4 represents methyl;
^R5 represents trifluoromethyl;
R ⁶ represents hydrogen, halogen, C _1-3 alkyl optionally substituted with 1 to 3 of the same or different halogens, or C _1-3 alkoxy optionally substituted with 1 to 3 of the same or different halogens;
Hy represents pyridin-3-yl.
or a pharmaceutically acceptable salt thereof,
A production method comprising the following steps 1 to 3:
(Step 1) Formula (4):
[In the formula, R ⁵ , R ⁶ , and Hy represent the same groups as above.]
or a salt thereof with a compound represented by formula (7):
[wherein R ⁴ represents the same group as above,
Pro represents tert-butoxycarbonyl;
A represents OH.
or a salt thereof in the presence of 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide and triethylamine to obtain a compound represented by the formula (8):
[In the formula, R ⁴ , R ⁵ , R ⁶ , Hy, and Pro represent the same groups as above.]
or a salt thereof;
(Step 2) The protecting group of the amino group of the compound represented by formula (8) or a salt thereof is deprotected with trifluoroacetic acid to obtain a compound represented by formula (9):
[In the formula, R ⁴ , R ⁵ , R ⁶ , and Hy represent the same groups as above.]
or a salt thereof;
(Step 3) A step of reacting a compound represented by formula (9) or a salt thereof with formaldehyde in the presence of sodium triacetoxyborohydride and acetic acid to produce a compound represented by formula (6) or a pharmaceutically acceptable salt thereof.

The present invention makes it possible to provide a compound represented by formula (1) or a pharmaceutically acceptable salt thereof. The compound or a pharmaceutically acceptable salt thereof inhibits or reduces the accumulation of abnormal protein aggregates in the brain, and is useful as a therapeutic or preventive agent for central nervous system diseases involving abnormal protein aggregates in the brain, particularly neurodegenerative diseases involving α-synuclein (Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Gaucher disease, infantile neuroaxonal dystrophy, etc.). Furthermore, by reproducing the spontaneous α-synuclein aggregates in neurons that are a pathological condition of Parkinson's disease, the compound is useful as an evaluation method for evaluating drugs that inhibit or reduce the accumulation of α-synuclein aggregates.

Figure 1 shows the difference in aggregate mass between neural spheroids derived from iPS cells of healthy individuals and neural spheroids derived from PLA2G6 mutant iPS cells. The vertical axis represents the aggregate mass within the neural spheroid, and the horizontal axis represents the number of days in culture. The white graph represents the aggregate mass of neural spheroids derived from healthy individuals, and the black graph represents the aggregate mass of neural spheroids derived from PLA2G6 mutant iPS cells. Figure 2 shows the difference in aggregate mass between dopamine neural spheroids derived from iPS cells of healthy individuals and dopamine neural spheroids derived from PLA2G6 mutant iPS cells. The vertical axis shows the aggregate mass in the dopamine neural spheroids, and the horizontal axis shows the number of days of culture. The white graph shows the aggregate mass of neural spheroids derived from healthy individuals, and the black graph shows the aggregate mass of neural spheroids derived from PLA2G6 mutant iPS cells. Figure 3 shows the difference in tyrosine hydroxylase levels between dopamine neural spheroids derived from iPS cells of healthy individuals and dopamine neural spheroids derived from PLA2G6 mutant iPS cells on day 26 of culture. The vertical axis shows the amount of tyrosine hydroxylase in the dopamine neural spheroids. The white graph shows the amount of tyrosine hydroxylase in neural spheroids derived from healthy individuals, and the black graph shows the amount of tyrosine hydroxylase in neural spheroids derived from PLA2G6 mutant iPS cells. Figure 4 shows the difference in the amount of cleaved caspase 3 in dopamine neural spheroids derived from iPS cells of healthy individuals and dopamine neural spheroids derived from PLA2G6 mutant iPS cells on day 40 of culture. The vertical axis shows the amount of cleaved caspase 3 in dopamine neural spheroids. The white graph shows the amount of cleaved caspase 3 in neural spheroids derived from healthy individuals, and the black graph shows the amount of cleaved caspase 3 in neural spheroids derived from PLA2G6 mutant iPS cells. Figure 5 shows the difference in aggregate mass between dopamine neural spheroids derived from iPS cells of healthy individuals and dopamine neural spheroids derived from iPS cells homozygous for the GBA1 gene mutation. The vertical axis shows the aggregate mass in the dopamine neural spheroids, and the horizontal axis shows the number of days of culture. The white graph shows the aggregate mass of neural spheroids derived from healthy individuals, and the black graph shows the aggregate mass of neural spheroids derived from iPS cells homozygous for the GBA1 gene mutation.

The present invention is described in detail below. In this specification, the number of carbon atoms in the definition of a "substituent" may be expressed as, for example, "C _1-3 ". Specifically, the expression "C _1-3 alkyl" is synonymous with alkyl having 1 to 3 carbon atoms.

Specific examples of "halogen" include fluorine, chlorine, bromine, and iodine. Fluorine or chlorine is preferred.

"C _1-3 alkyl" means a linear or branched saturated hydrocarbon group having 1 to 3 carbon atoms. Preferably, it is a "C _1-2 alkyl". Specific examples of "C _1-3 alkyl" include methyl, ethyl, propyl, isopropyl, etc.

The "C _1-3 alkyl" part of "C _1-3 alkoxy" has the same meaning as the above-mentioned "C _1-3 alkyl". Preferably, it is "C _1-2 alkoxy". Specific examples of "C _1-3 alkoxy" include methoxy, ethoxy, propoxy, isopropoxy, etc.

Among the compounds of the present invention represented by formula (1), preferred X, Y, m, n, r, s, ^R1 , ^R2 , ^R3 , ^R4 , ^R5 , ^R6 and ^R7 are as follows, but the technical scope of the present invention is not limited to the range of the compounds listed below.

In the compound represented by formula (1), R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ may be substituted on any carbon atom if substitution is possible, and R ¹ and R ² , and R ³ and R ⁴ may be substituted on the same carbon atom if substitution is possible, and R ³ and R ⁴ may be substituted with H of CH when Y is CH.
In the compound represented by formula (1), the binding site (arrow) of Hy where Hy is bound to the nitrogen-containing saturated heterocycle is carbon.

Preferably, X is oxygen, NH, or NMe, and more preferably, oxygen or NMe.

Y is preferably CH.

n is preferably 1.

Preferably, m is 1.

When Y is CH, r is preferably 0, 1 or 2, more preferably 0 or 1, and even more preferably 0.
When Y is nitrogen, r is 1, 2, 3 or 4, with 1 being preferred.

When Y is CH, s is preferably 0, 1, or 2, more preferably 1 or 2, and even more preferably 1. When Y is nitrogen, s is 2.

R ¹ is preferably hydrogen, methyl or fluorine, more preferably hydrogen.

^R2 is preferably hydrogen, methyl or fluorine, more preferably hydrogen.

^R3 is preferably hydrogen, methyl or ethyl, more preferably hydrogen.

^R4 is preferably hydrogen, methyl or ethyl, more preferably methyl or ethyl, and even more preferably methyl.

In a ring containing X and Y, an example of a structure in which ^R3 and ^R4 combine to form a bridged methylene or ethylene includes the ring containing X and Y and the structure of the following formula (3). In the following formula (3), the wavy line indicates the bonding site to the carbonyl in formula (1). In the following formula (3), X and Y have the same meanings as in item 1.

^R5 is preferably halogen or C _1-3 alkyl optionally substituted with 1 to 3 halogen atoms which may be the same or different, more preferably halogen or methyl optionally substituted with 1 to 3 fluorine atoms, even more preferably trifluoromethyl, methyl or fluorine, and most preferably trifluoromethyl.

^R6 is preferably hydrogen, halogen, or C _1-3 alkyl optionally substituted with 1 to 3 halogen atoms which may be the same or different, more preferably hydrogen, halogen, or methyl optionally substituted with 1 to 3 fluorine atoms, even more preferably hydrogen, halogen, or methyl, and most preferably hydrogen, fluorine, or methyl.

^R7 is preferably hydrogen or _C1-3 alkyl, more preferably hydrogen or methyl, and even more preferably methyl. Another embodiment of ^R7 is _C1-3 alkyl containing 1 to 5 deuterium atoms.

Hy is preferably a pyridine ring, more preferably pyridin-3-yl represented by the following formula (4): In the following formula (4), the arrow indicates the bonding position to the nitrogen-containing saturated ring.

Condensing agents used in the condensation reaction include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide, etc. Preferred are 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, and 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide.

Reducing agents include sodium borohydride, sodium triacetoxyborohydride, sodium cyanoborohydride, etc. Preferred are sodium cyanoborohydride and sodium triacetoxyborohydride.

Examples of formaldehyde or its equivalents include formaldehyde, 1,3,5-trioxane, and paraformaldehyde. Formaldehyde is preferred.

Protective groups for amino groups include tert-butoxycarbonyl and benzyloxycarbonyl groups, with tert-butoxycarbonyl being preferred.

Preferred compounds among those represented by formula (1) include the following compounds or pharmaceutically acceptable salts thereof:

One embodiment of the compound represented by formula (1) is the following (A).
(A)
X is oxygen or ^NR7 ;
R ⁷ is hydrogen, C _1-3 alkyl or cyclopropyl;
Y is CH or nitrogen;
m is 0, 1, or 2;
n is 0 or 1;
r is 0, 1 or 2;
s is 0, 1 or 2;
(wherein, when s=0, Y is CH and r is 1 or 2;
when s=1, Y is CH and r is 0, 1 or 2;
When s=2, r is 1 or 2.
R ¹ is hydrogen, halogen, methyl or hydroxy;
^R2 is hydrogen, halogen, methyl or hydroxy;
R ³ is hydrogen or C _1-3 alkyl;
R ⁴ is hydrogen or C _1-3 alkyl;
wherein ^R3 and ^R4 together may form a bridged methylene or ethylene;
R ⁵ is halogen or C _1-3 alkyl optionally substituted with 1 to 3 of the same or different halogens;
R ⁶ is hydrogen, halogen, or C _1-3 alkyl optionally substituted with 1 to 3 of the same or different halogens;
Hy is a pyridine ring, a pyridazine ring, a pyrimidine ring, or a pyrazine ring;
Here,
(I) When Hy including ^R5 and ^R6 is 5-fluoropyridin-2-yl, and m and n are 1, r is 0 and s is 1;
(II) When ^R5 is methyl and ^R6 is hydrogen, r is 0 and s is 1;
however,
(I') Hy including ^R5 and ^R6 is not 4,6-dimethylpyrimidin-2-yl or 5-bromopyrimidin-2-yl;
(II') When Hy including ^R5 and ^R6 is 5-chloropyridin-2-yl, m and n are 1, and ^R1 and ^R2 are not both hydrogen;
However, the following compound (III') (3-ethoxyoxetan-3-yl)[3-{4-(trifluoromethyl)pyrimidin-2-yl}pyrrolidin-1-yl]methanone,
or a pharmaceutically acceptable salt thereof.

One embodiment of the compound represented by formula (1) is the following (B).
(B)
X is oxygen or ^NR7 ;
R ⁷ is hydrogen, C _1-3 alkyl, or cyclopropyl;
Y is CH or nitrogen;
m is 0, 1, or 2;
n is 0 or 1;
r is 0, 1 or 2;
s is 0, 1 or 2;
(wherein, when s=0, Y is CH and r is 1 or 2;
when s=1, Y is CH and r is 0, 1 or 2;
When s=2, r is 1 or 2.
R ¹ is hydrogen, halogen, methyl or hydroxy;
^R2 is hydrogen, halogen, methyl or hydroxy;
R ³ is hydrogen or C _1-3 alkyl;
R ⁴ is hydrogen or C _1-3 alkyl;
wherein ^R3 and ^R4 together may form a bridged methylene or ethylene;
R ⁵ is halogen or C _1-3 alkyl optionally substituted with 1 to 3 of the same or different halogens;
R ⁶ is hydrogen, halogen, or C _1-3 alkyl optionally substituted with 1 to 3 of the same or different halogens;
Hy is a pyridine ring;
Here,
(I) When Hy including ^R5 and ^R6 is 5-fluoropyridin-2-yl and m and n are 1, r is 0 and s is 1;
(II) When ^R5 is methyl and ^R6 is hydrogen, r is 0 and s is 1;
however,
(I') A compound or a pharmaceutically acceptable salt thereof, wherein when Hy including ^R5 and ^R6 is 5-chloropyridin-2-yl, m and n are 1, and ^R1 and ^R2 are not both hydrogen.

One embodiment of the compound represented by formula (1) is the following (C).
(C)
X is oxygen or ^NR7 ;
R ⁷ is hydrogen, C _1-3 alkyl or cyclopropyl;
Y is CH;
m is 1,
n is 1,
r is 0, 1 or 2;
s is 0, 1 or 2;
(However, when s=0, r is 1 or 2,
When s=1, r is 0, 1 or 2;
When s=2, r is 1 or 2.
R ¹ is hydrogen, methyl or fluorine;
^R2 is hydrogen, methyl or fluorine;
^R3 is hydrogen, methyl or ethyl;
^R4 is hydrogen, methyl or ethyl;
R ⁵ is halogen or C _1-3 alkyl optionally substituted with 1 to 3 of the same or different halogens;
R ⁶ is hydrogen, halogen, or C _1-3 alkyl optionally substituted with 1 to 3 of the same or different halogens;
Hy is a pyridine ring;
Here,
(I) When Hy including ^R5 and ^R6 is 5-fluoropyridin-2-yl, r is 0 and s is 1;
(II) When ^R5 is methyl and ^R6 is hydrogen, r is 0 and s is 1;
however,
(I') A compound or a pharmaceutically acceptable salt thereof, wherein when Hy including ^R5 and ^R6 is 5-chloropyridin-2-yl, ^R1 and ^R2 are not both hydrogen.

One embodiment of the compound represented by formula (1) is the following (D).
(D)
X is oxygen or ^NR7 ;
^R7 is hydrogen or methyl;
Y is CH;
m is 1,
n is 1,
r is 0 or 1;
s is 1 or 2;
(However, when s = 2, r is 1.)
^R1 is hydrogen;
^R2 is hydrogen;
^R3 is hydrogen;
^R4 is methyl or ethyl;
R ⁵ is halogen or methyl optionally substituted with 1 to 3 fluorines;
R ⁶ is hydrogen, halogen, or methyl optionally substituted with 1 to 3 fluorines;
Hy is pyridin-3-yl;
or a pharmaceutically acceptable salt thereof.

One embodiment of the compound represented by formula (1) is the following (E), which is a structure of formula (2).
(E)
In formula (2),
X is oxygen or NMe;
^R4 is methyl;
^R5 is trifluoromethyl, methyl or fluorine;
^R6 is hydrogen, halogen or methyl;
Hy is pyridin-3-yl;
or a pharmaceutically acceptable salt thereof.

One embodiment of the compound represented by formula (1) is the following compound or a pharmaceutically acceptable salt thereof:
(3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Example 1),
(3-methyloxetan-3-yl){4-[5-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Example 2),
(3-methyloxetan-3-yl){4-[4-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Example 3),
(3-methyloxetan-3-yl){4-[2-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Example 4),
{4-[5-fluoro-6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}(3-methyloxetan-3-yl)methanone (Example 29),
(3-methyloxetan-3-yl){4-[4-methyl-6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Example 31),
(1,3-dimethylazetidin-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Example 41),
(1,3-dimethylazetidin-3-yl){4-[2-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Example 42),
(1,3-Dimethylazetidin-3-yl){4-[4-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Example 43), or (1,3-dimethylazetidin-3-yl){4-[5-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Example 44).

One embodiment of the compound represented by formula (1) is the following compound or a pharmaceutically acceptable salt thereof:
(3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Example 1),
(3-Methyloxetan-3-yl){4-[5-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Example 2)
{4-[5-fluoro-6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}(3-methyloxetan-3-yl)methanone (Example 29),
(3-methyloxetan-3-yl){4-[4-methyl-6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Example 31),
(1,3-Dimethylazetidin-3-yl){4-[2-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Example 42), or (1,3-dimethylazetidin-3-yl){4-[5-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone (Example 44).

In the present invention, "pharmaceutically acceptable salts" include acid addition salts and base addition salts. Examples of acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, and phosphate, and organic acid salts such as citrate, oxalate, phthalate, fumarate, maleate, succinate, malate, acetate, formate, propionate, benzoate, trifluoroacetate, methanesulfonate, benzenesulfonate, para-toluenesulfonate, and camphorsulfonate. Examples of base addition salts include inorganic base salts such as sodium salt, potassium salt, calcium salt, magnesium salt, barium salt, and aluminum salt, as well as salts with organic bases such as trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, tromethamine [tris(hydroxymethyl)methylamine], tert-butylamine, cyclohexylamine, dicyclohexylamine, and N,N-dibenzylethylamine. Furthermore, "pharmaceutically acceptable salts" also include amino acid salts with basic or acidic amino acids such as arginine, lysine, ornithine, aspartic acid, and glutamic acid.

Suitable salts of starting compounds and intermediates, and salts acceptable as pharmaceutical raw materials, are conventional non-toxic salts, including acid addition salts such as organic acid salts (e.g., acetate, trifluoroacetate, maleate, fumarate, citrate, tartrate, methanesulfonate, benzenesulfonate, formate, para-toluenesulfonate, etc.) and inorganic acid salts (e.g., hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate, etc.); salts with amino acids (e.g., arginine, aspartic acid, glutamic acid, etc.); metal salts such as alkali metal salts (e.g., sodium salt, potassium salt, etc.) and alkaline earth metal salts (e.g., calcium salt, magnesium salt, etc.); ammonium salts; or organic base salts (e.g., trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, etc.), as well as other salts that can be appropriately selected by those skilled in the art.

When a salt of a compound of the present invention is desired, if the compound is obtained in the form of a salt, it can be purified as is. Alternatively, if the compound is obtained in the free form, it can be dissolved or suspended in an appropriate organic solvent, and an acid or base can be added to form a salt by a commonly used method.

In the present invention, deuterium-converted compounds in which one or more ¹ H in a compound represented by formula (1) is converted to ² H(D) are also encompassed by the compound represented by formula (1). The present invention includes a compound represented by formula (1) or a pharmaceutically acceptable salt thereof. In addition, the compound of the present invention may exist in the form of a hydrate and/or a solvate with various solvents (such as an ethanol solvate), and these hydrates and/or solvates are also included in the compound of the present invention. Furthermore, the present invention includes all tautomers, all existing stereoisomers, and all crystalline forms of compound (1) of the present invention, as well as mixtures thereof.

The compounds of the present invention may exist as optical isomers based on optically active centers, atropisomers based on axial or planar chirality caused by restricted intramolecular rotation, other stereoisomers, tautomers, and geometric isomers. All possible isomers, including these, and mixtures thereof, are also included in the compounds of the present invention.

In particular, optical isomers and atropisomers can be obtained as racemates or, when optically active starting materials or intermediates are used, as optically active isomers. If necessary, at an appropriate stage in the production methods described below, the corresponding racemates of the starting materials, intermediates, or final products can be physically or chemically resolved into their optical antipodes by known separation methods, such as methods using optically active columns or fractional crystallization. Specifically, for example, in the diastereomeric method, two diastereomers are formed from a racemate by reaction with an optically active resolving agent. These different diastereomers generally have different physical properties and can be resolved by known methods, such as fractional crystallization.

The following describes an example of a method for producing the compound represented by formula (1) of the present invention, but the present invention is not limited to this example.

Production Method The compounds of the present invention are synthesized by the following production method and a method combining known synthesis methods.
The compounds in the reaction schemes may each form a salt, and examples of such salts include the same as the salts of the compound represented by formula (1). Note that these reactions are merely illustrative, and the compound of the present invention may also be produced by other appropriate methods based on the knowledge of a person skilled in organic synthesis.

In each of the production methods described below, even if the use of a protecting group is not specifically specified, if a functional group that requires protection is present, the functional group may be protected as necessary, and the desired product may be obtained by deprotecting it after the reaction or a series of reactions have been completed.

Introduction and removal of a protecting group can be carried out by a method commonly used in organic synthetic chemistry (e.g., the method described in T.W. Greene and P.G.M. Wuts, "Protective Groups in Organic Synthesis", 3rd Ed., John Wiley and Sons, Inc., New York (1999)) or a method similar thereto.
Examples of the amino-protecting group include tert-butoxycarbonyl, benzyloxycarbonyl, p-toluenesulfonyl, o-nitrobenzenesulfonyl, 4-methoxybenzyl, 2,4-dimethoxybenzyl, and the like.

Manufacturing method 1
Among the compounds represented by formula (1), the compound represented by formula (1a) is produced, for example, by the method shown below.
[In the formula, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , m, n, r, s, Y, and Hy are as defined in [Item 1] above; and A represents halogen or OH.]

(Step 1-1: Production process of compound (1a))
Compound (1a) is produced by reacting compound (1-1) with compound (1-2) in a suitable inert solvent in the presence or absence of various condensing agents and/or bases. Compound (1-1) may be a commercially available compound or one produced by known methods (e.g., International Publication No. WO 2014/192868). Compound (1-1) may also be one produced by Production Methods 3 to 6 described below. Compound (1-2) may be a commercially available compound or one produced by known methods (e.g., International Publication No. WO 2016/004272). The base used in this step is appropriately selected from the bases exemplified below, and examples include sodium hydride, triethylamine, diisopropylethylamine, and sodium carbonate. The condensing agent used in this step can be any of a variety of condensing agents commonly used in organic synthesis reactions, including 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, and 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide. The solvent used in this step can be appropriately selected from the solvents exemplified below, including DMF, THF, dichloromethane, chloroform, and ethyl acetate. The reaction time in this step is typically 5 minutes to 72 hours, preferably 30 minutes to 24 hours. The reaction temperature in this step is typically −78°C to 200°C, preferably −78°C to 80°C.

Manufacturing method 2
Among the compounds represented by formula (1), the compounds represented by formula (1b) and formula (1c) are produced, for example, by the method shown below.
[In the formula, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , m, n, r, s, Y, and Hy are as defined in [Item 1] above; R ⁸ represents C _1-3 alkyl or cyclopropyl optionally substituted with 1 to 3 halogen atoms, which may be the same or different; A represents halogen or OH; and Pro represents an amino-protecting group.]

(Step 2-1: Production step of compound (1c))
Compound (1c) is produced from compound (1-1) and compound (2-1) according to the method described in step 1-1. Compound (2-1) may be a commercially available compound or one produced by a known method (e.g., International Publication No. WO2010/026096).

(Step 2-2: Production step of compound (2-3))
Compound (2-3) is produced from compound (1-1) and compound (2-2) according to the method described in step 1-1. Compound (2-2) may be a commercially available compound or one produced by a known method (e.g., International Publication No. WO2008/085117).

(Step 2-3: Production step of compound (1b))
Compound (1b) is produced by deprotecting the amino-protecting group Pro of compound (2-3) by a known method (for example, the method described in Protective Groups in Organic Synthesis, 3rd Edition (Theodora W. Green and Peter G.M. Wuts, published by John Wiley & Sons Inc., 1999)). Examples of the amino-substituent Pro include a tert-butoxycarbonyl group and a benzyloxycarbonyl group.

(Step 2-4: Production step of compound (1c))
Compound (1c) is produced by reacting compound (1b) with various aldehydes, ketones, ketone equivalents, etc. corresponding to ^R8 in a suitable inert solvent in the presence of a reducing agent. Various reducing agents commonly used in organic synthesis reactions can be used as the reducing agent, including sodium borohydride, sodium triacetoxyborohydride, sodium cyanoborohydride, etc. The solvent used in this step is appropriately selected from the solvents exemplified below, including toluene, THF, dichloromethane, methanol, etc. The reaction time is usually 5 minutes to 48 hours, preferably 1 hour to 24 hours. The reaction temperature is usually −78°C to 100°C, preferably 0°C to 80°C.

Compound (1c) can also be produced by reacting compound (1b) with various alkyl halides or alkyl sulfonates corresponding to ^R8 in an appropriate inert solvent in the presence of a base. The base is appropriately selected from the bases exemplified below, and examples thereof include potassium carbonate, cesium carbonate, sodium hydride, and lithium diisopropylamide. The solvent used in this step is appropriately selected from the solvents exemplified below, and examples thereof include DMF, dimethyl sulfoxide, THF, and 1,4-dioxane. The reaction time is usually 5 minutes to 48 hours, and preferably 1 hour to 24 hours. The reaction temperature is usually −78° C. to 100° C., and preferably 0° C. to 80° C.

Manufacturing method 3
The compound represented by formula (1-1) can be produced, for example, by the method shown below.
[In the formula, R ¹ , R ² , R ⁵ , R ⁶ , m, n, and Hy are as defined in [Item 1] above; W ¹ and W ² represent halogen; and Pro represents an amino-protecting group.]

(Step 3-1: Production step of compound (3-3))
Compound (3-3) is produced by reacting compound (3-1) with compound (3-2) in a suitable inert solvent in the presence of zinc and a palladium catalyst. Compound (3-1) may be a commercially available compound or one produced by a known method (e.g., International Publication No. WO2008/147831). Compound (3-2) may be a commercially available compound or one produced by a known method (e.g., Bioorganic & Medicinal Chemistry Letters (2006), 16(17), 4528-4532). Examples of halogens in ^W1 and ^W2 include chlorine, bromine, and iodine. Examples of the amino group substituent Pro include a tert-butoxycarbonyl group and a benzyloxycarbonyl group. Examples of palladium catalysts that can be used include various palladium catalysts commonly used in conventional methods, such as tetrakis(triphenylphosphine)palladium(0). The solvent used in this step is appropriately selected from the solvents exemplified below, and examples thereof include DMF, dimethylacetamide, etc. The reaction time is usually 5 minutes to 48 hours, preferably 1 hour to 24 hours. The reaction temperature is usually 0°C to 100°C, preferably 0°C to 80°C.

(Step 3-2: Production step of compound (1-1))
The compound (1-1) is produced from the compound (3-3) according to the method described in step 2-3.

Manufacturing method 4
Among the compounds represented by formula (1-1), the compound represented by formula (1-1a) can be produced, for example, by the method shown below.
[In the formula, R ¹ , R ² , R ⁵ , R ⁶ , n, and Hy are as defined in [Item 1] above; W ² represents a halogen; Pro represents an amino-protecting group; and T represents a boronic acid or a boronic acid ester.]

(Step 4-1: Production step of compound (4-2))
Compound (4-2) is produced by reacting compound (4-1) with compound (3-2) in a suitable inert solvent in the presence of a palladium catalyst. This step can be carried out in the presence of a base and/or a phosphorus ligand, as necessary. Compound (4-1) may be a commercially available compound or one produced by a known method (e.g., International Publication No. WO 2019/163865 ). Examples of the amino group substituent Pro include a tert-butoxycarbonyl group and a benzyloxycarbonyl group. Various palladium catalysts commonly used in conventional methods can be used as the palladium catalyst, including, for example, tetrakis(triphenylphosphine)palladium(0). The base used in this step can be appropriately selected from the bases exemplified below, including, for example, potassium carbonate and cesium carbonate. The phosphorus ligand used in this step can be any of various phosphorus ligands commonly used in organic synthesis reactions, including, for example, triphenylphosphine and bis(diphenylphosphino)methane. The solvent used in this step is appropriately selected from the solvents exemplified below, and examples include 1,4-dioxane, tetrahydrofuran, water, and mixed solvents thereof. The reaction temperature is usually 0°C to 200°C, preferably 20°C to 150°C, and the reaction can be performed under microwave irradiation, if necessary. The reaction time varies depending on conditions such as the reaction temperature, the palladium catalyst used, the raw materials, and the solvent, but is usually 5 minutes to 72 hours, and preferably 1 hour to 24 hours.

(Step 4-2: Production step of compound (4-3))
Compound (4-3) is produced by reacting compound (4-2) in a suitable inert solvent in the presence of a catalyst under a hydrogen atmosphere. The solvent used in this step is appropriately selected from the solvents exemplified below, and examples thereof include methanol, ethanol, chloroform, and mixed solvents thereof. As the catalyst, various catalysts commonly used in catalytic reduction reactions can be used, and examples thereof include palladium carbon and palladium hydroxide. The reaction time is usually 5 minutes to 48 hours, preferably 1 hour to 24 hours. The reaction temperature is usually 0°C to 100°C, preferably 0°C to 40°C.

(Step 4-3: Production step of compound (1-1a))
The compound (1-1a) is produced from the compound (4-3) according to the method described in step 2-3.

Manufacturing method 5
Among the compounds represented by formula (1-1), the compound represented by formula (1-1b) can be produced, for example, by the method shown below.
[In the formula, R ² , R ⁵ , R ⁶ , m, n, and Hy are as defined in [Item 1] above; W ² represents a halogen; and Pro represents an amino-protecting group.]

(Step 5-1: Production step of compound (5-2))
Compound (5-2) is produced by reacting compound (3-2) with compound (5-1) in the presence of an alkyllithium in a suitable inert solvent. Compound (5-1) may be a commercially available compound or one produced by a known method (e.g., International Publication No. WO 2005/058888). Examples of the amino group substituent Pro include a tert-butoxycarbonyl group and a benzyloxycarbonyl group. Examples of alkyllithium include various alkyllithiums commonly used in conventional methods, such as butyllithium. The solvent used in this step is appropriately selected from the solvents exemplified below, including tetrahydrofuran and diethyl ether. The reaction time is typically 5 minutes to 48 hours, preferably 1 hour to 24 hours. The reaction temperature is typically −78°C to 100°C, preferably −78°C to 40°C.

(Step 5-2: Production step of compound (1-1b))
Compound (1-1b) is produced from compound (5-2) according to the method described in step 2-3.

Manufacturing method 6
Among the compounds represented by formula (1-1), the compound represented by formula (1-1c) can be produced, for example, by the method shown below.
[In the formula, R ² , R ⁵ , R ⁶ , m, n, and Hy are as defined in the above [Item 1]; Q represents a halogen; and Pro represents an amino-protecting group.]

(Step 6-1: Production step of compound (6-1))
Compound (6-1) is produced by reacting compound (5-2) in the presence of a halogenating agent in an appropriate inert solvent. Examples of the amino group substituent Pro include a tert-butoxycarbonyl group and a benzyloxycarbonyl group. Examples of the halogen in Q include fluorine and chlorine. Various halogenating agents commonly used in conventional methods can be used as the halogenating agent, including (diethylamino)sulfur trifluoride, bis(2-methoxyethyl)aminosulfur trifluoride, and phosphorus oxychloride. The solvent used in this step is appropriately selected from the solvents exemplified below, including dichloromethane, chloroform, 1,4-dioxane, and toluene. The reaction time is generally 5 minutes to 48 hours, preferably 1 hour to 24 hours. The reaction temperature is generally −78°C to 100°C, preferably −78°C to 40°C.

(Step 6-2: Production step of compound (1-1c))
Compound (1-1c) is produced from compound (6-1) according to the method described in step 2-3.

Manufacturing method 7
Among the compounds represented by formula (1), the compound represented by formula (1d) is produced, for example, by the method shown below.
[In the formula, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , m, n, r, s, and Hy are the same as defined in [Item 1] above.]

(Step 7-1: Production step of compound (1d))
Compound (1d) is produced by reacting compound (7-1) with triphosgene and a base in an appropriate inert solvent, followed by reaction with compound (1-1). The base used in this step is appropriately selected from the bases exemplified below, and examples thereof include pyridine and triethylamine. The solvent used in this step is appropriately selected from the solvents exemplified below, and examples thereof include dichloromethane and chloroform. The reaction time is usually 5 minutes to 48 hours, and preferably 30 minutes to 24 hours. The reaction temperature is usually −78° C. to 100° C., and preferably 0° C. to 80° C.

Manufacturing method 8
Among the compounds represented by formula (1), the compound represented by formula (1e) is produced, for example, by the method shown below.
[In the formula, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , m, n, r, s, and Hy are as defined in [Item 1] above; and R ⁸ represents C _1-3 alkyl or cyclopropyl optionally substituted with 1 to 3 halogen atoms, which may be the same or different.]
(Step 8-1: Production step of compound (1e))
Compound (1e) is produced from compound (8-1) and compound (1-1) according to the method described in step 7-1.

Manufacturing method 9
Among the compounds represented by formula (1), the compounds represented by formula (1e) and formula (1f) are produced, for example, by the method shown below.
[In the formula, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , m, n, r, s, and Hy are as defined in [Item 1] above; R ⁸ represents C _1-3 alkyl or cyclopropyl optionally substituted with 1 to 3 halogen atoms, which may be the same or different; and Pro represents an amino-protecting group.]

(Step 9-1: Production step of compound (9-2))
Compound (9-2) is produced from compound (9-1) and compound (1-1) according to the method described in step 7-1. Examples of the amino group substituent Pro include a tert-butoxycarbonyl group and a benzyloxycarbonyl group.

(Step 9-2: Production step of compound (1f))
Compound (1f) is produced from compound (9-2) according to the method described in step 2-3.

(Step 9-3: Production step of compound (1e))
Compound (1e) is produced from compound (1f) according to the method described in step 2-4.

Among the raw materials or intermediates used in the manufacturing methods described above, those for which no particular manufacturing method is specifically described are commercially available compounds or can be synthesized from commercially available compounds by methods known to those skilled in the art or by methods similar thereto.

The base used in each step of the above-mentioned production methods should be selected appropriately depending on the reaction and the type of raw material compound, etc., but examples include alkali bicarbonates such as sodium bicarbonate and potassium bicarbonate; alkali carbonates such as sodium carbonate and potassium carbonate; metal hydrides such as sodium hydride and potassium hydride; alkali metal hydroxides such as sodium hydroxide and potassium hydroxide; alkali metal alkoxides such as sodium methoxide and sodium t-butoxide; organometallic bases such as butyllithium and lithium diisopropylamide; and organic bases such as triethylamine, diisopropylethylamine, pyridine, 4-dimethylaminopyridine (DMAP), and 1,8-diazabicyclo[5.4.0]-7-undecene (DBU).

The solvent used in each step of the above-described production methods should be selected appropriately depending on the reaction and the type of raw material compounds, but examples include alcohols such as methanol, ethanol, and isopropanol; ketones such as acetone and methyl ketone; halogenated hydrocarbons such as methylene chloride and chloroform; ethers such as tetrahydrofuran (THF) and dioxane; aromatic hydrocarbons such as toluene and benzene; aliphatic hydrocarbons such as hexane and heptane; esters such as ethyl acetate and propyl acetate; amides such as N,N-dimethylformamide (DMF) and N-methyl-2-pyrrolidone (NMP); sulfoxides such as dimethyl sulfoxide (DMSO); and nitriles such as acetonitrile. These solvents can be used alone or in combination. Depending on the type of reaction, organic bases such as diazabicycloundecene (DBU) may also be used as solvents.

The compound of the present invention represented by formula (1) or an intermediate thereof can be separated or purified by methods known to those skilled in the art, such as extraction, partitioning, reprecipitation, column chromatography (e.g., silica gel column chromatography, ion exchange column chromatography, or preparative liquid chromatography), or recrystallization.
Examples of recrystallization solvents that can be used include alcoholic solvents such as methanol, ethanol, and 2-propanol; ether solvents such as diethyl ether; ester solvents such as ethyl acetate; aromatic hydrocarbon solvents such as benzene and toluene; ketone solvents such as acetone; halogenated solvents such as dichloromethane and chloroform; hydrocarbon solvents such as hexane; aprotic solvents such as dimethylformamide and acetonitrile; water; and mixtures of these solvents. Other purification methods that can be used include those described in Volume 1 of "Experimental Chemistry Lectures" (edited by the Chemical Society of Japan, Maruzen). Furthermore, the molecular structure of the compound of the present invention can be easily determined by spectroscopic techniques such as nuclear magnetic resonance, infrared absorption, and circular dichroism spectroscopy, and/or mass spectrometry, with reference to the structures derived from the respective starting compounds.

Furthermore, intermediates or final products in the above-mentioned production methods can be converted into other compounds within the scope of the present invention by appropriately converting their functional groups, particularly by extending various side chains using amino, hydroxyl, carbonyl, halogen, etc. as a foothold, and by performing the above-mentioned protection and deprotection as necessary. Functional group conversion and side chain extension can be carried out using commonly used methods (see, for example, Comprehensive Organic Transformations, R. C. Larock, John Wiley & Sons Inc. (1999)).

The compounds of the present invention represented by formula (1) may have asymmetry or may contain a substituent with an asymmetric carbon, and such compounds may have optical isomers. The compounds of the present invention include mixtures of these isomers or isolated isomers, and can be produced according to conventional methods. Production methods include, for example, methods using raw materials with an asymmetric center or methods introducing asymmetry at an intermediate stage. For example, in the case of optical isomers, optical isomers can be obtained by using optically active raw materials or by performing optical resolution at an appropriate stage in the production process. For example, when the compound represented by formula (1) or an intermediate thereof has a basic functional group, optical resolution can be achieved by a diastereomeric method in which a salt is formed using an optically active acid (e.g., monocarboxylic acids such as mandelic acid, N-benzyloxyalanine, and lactic acid; dicarboxylic acids such as tartaric acid, o-diisopropylidenetartaric acid, and malic acid; sulfonic acids such as camphorsulfonic acid and bromocamphorsulfonic acid) in an inert solvent (e.g., alcoholic solvents such as methanol, ethanol, and 2-propanol; ether solvents such as diethyl ether; ester solvents such as ethyl acetate; hydrocarbon solvents such as toluene; aprotic solvents such as acetonitrile; or a mixed solvent of two or more of the above solvents). When the compound represented by formula (1) of the present invention or an intermediate thereof has an acidic functional group such as a carboxyl group, optical resolution can also be achieved by forming a salt using an optically active amine (e.g., organic amines such as 1-phenylethylamine, quinine, quinidine, cinchonidine, cinchonine, and strychnine).

The temperature for salt formation is selected from the range of -50°C to the boiling point of the solvent, preferably from 0°C to the boiling point, and more preferably from room temperature to the boiling point of the solvent. To improve optical purity, it is desirable to first raise the temperature to near the boiling point of the solvent. When filtering the precipitated salt, cooling can be performed as needed to improve yield. The amount of optically active acid or amine used is approximately 0.5 to approximately 2.0 equivalents relative to the substrate, preferably approximately 1 equivalent. If necessary, the crystals can be recrystallized in an inert solvent (e.g., alcoholic solvents such as methanol, ethanol, and 2-propanol; etheric solvents such as diethyl ether; esteric solvents such as ethyl acetate; hydrocarbon solvents such as toluene; aprotic solvents such as acetonitrile; or a mixture of two or more of the above solvents) to obtain a highly pure optically active salt. Furthermore, if necessary, the optically resolved salt can be treated with an acid or base using conventional methods to obtain the free form.

In Lewy body diseases such as Parkinson's disease, abnormally aggregated α-synuclein is found in the brains of patients. Therefore, the drug of this application, which inhibits or reduces the accumulation of α-synuclein aggregates, is expected to be effective in improving the pathology of these diseases.

Furthermore, these aggregates are thought to exhibit neurotoxicity, inducing neuronal vulnerability and neuronal cell death, and contributing to the onset and progression of the disease. Therefore, the drug of the present application, which suppresses the neurotoxicity and neuronal cell death associated with α-synuclein aggregates, is expected to be effective in improving the pathology of Lewy body diseases such as Parkinson's disease.

The production of neurotransmitters is one of the neuronal functions, and a decrease in neurotransmitters indicates neuronal vulnerability. For example, in dopaminergic neurons, neuronal vulnerability is indicated by a decrease in the amount of tyrosine hydroxylase, which is involved in dopamine metabolism.

Furthermore, electroencephalogram (EEG) abnormalities have been reported in Lewy body diseases such as Parkinson's disease. EEG is a manifestation of synchronized neural activity. Therefore, the drug of this application, which normalizes synchronized neural activity associated with α-synuclein aggregates, is expected to have an ameliorative effect on the pathology of these diseases.

Neural spheroids used to measure the amount of α-synuclein aggregates can be prepared, for example, by three-dimensionally culturing neural stem cells or dopamine (DA) neural progenitor cells prepared from human iPS cells mutated in a synucleopathy-associated gene under neuronal differentiation induction. The amount of α-synuclein aggregates can be evaluated by measuring the amount of high-molecular-weight α-synuclein using three-dimensionally cultured neural spheroids by a protein analysis technique using an α-synuclein antibody.
Furthermore, synchronous neural firing can be evaluated by performing imaging analysis using three-dimensionally cultured neural spheroids with a fluorescent calcium probe.
Furthermore, by using the steps of measuring the amount of α-synuclein aggregates and measuring synchronized neuronal firing in neural spheroids, it is possible to reproduce the pathology of Parkinson's disease, and to evaluate drugs that have the effect of inhibiting or reducing the accumulation of α-synuclein aggregates in the pathology of Parkinson's disease.

Differentiation of synucleopathy-associated gene-mutated human iPS cells into neural stem cells can be induced, for example, by using PLA2G6 gene-mutated cells established from an iPS cell line derived from a healthy individual (clone name 201B7, obtained from the Center for iPS Cell Research and Application, Kyoto University), culturing them in StemFitAK03N medium (Ajinomoto Co., Basic03) at 37°C and 5% _CO2 , and then inducing differentiation using PSC Neuronal Induction Medium (Thermo Fisher Scientific, cat#A1647801).

As a culture medium for neural stem cells, for example, one having the following composition can be used.
<Composition of neural stem cell culture medium>
Neurobasal medium (Thermo Fisher Scientific 2113049)
Advanced DMEM/F-12 medium (Thermo Fisher Scientific, 12634028)
Neural Induction Supplement (Thermo Fisher Scientific A1647801)

Neural stem cells can be induced to differentiate into neural spheroids, for example, by seeding neural stem cells (10,000 cells/well) in a 96-well U-shaped plate (Thermo Fisher Scientific, cat. #174929) and culturing them in culture medium at 37°C and 5% _CO2 , with half of the medium replaced on days 2 and 4 after differentiation induction.

As a differentiation medium for neural spheroids of neural stem cells, for example, a medium having the following composition can be used.
<Culture medium composition for neural spheroids>
BrainPhys Neuronal Medium (manufactured by STEMCELL Technologies, cat#ST-05793)
NeuroCult SM1 Neuronal Supplement (manufactured by STEMCELL Technologies, cat#05711)
N2 Supplement-A (STEMCELL Technologies, cat#07152)
20 ng/mL BDNF (Peprotech, cat#450-02)
20 ng/mL GDNF (Peprotech, cat#450-10)
1 mM dibutyryl cAMP (Nacalai, cat#11540-74)
200 nM ascorbic acid (Nacalai, cat#03420-52)

Dopaminergic neural progenitor cells can be differentiated from human iPS cells mutated in a synucleopathy-associated gene by, for example, using a dopaminergic neural induction kit (Thermo Fisher Scientific, cat#A3147701) to induce dopaminergic neural progenitor cells from PLA2G6 gene mutant cells or GBA1 gene homozygous mutant cells established from an iPS cell line derived from a healthy individual.

Differentiation of dopaminergic neural progenitor cells into neural spheroids can be induced, for example, by culturing cryopreserved dopaminergic neural progenitor cells at 37°C and 5% _CO2 using a Floor Plate Cell Expansion Kit (Thermo Fisher Scientific, cat#A3165801), seeding the resulting dopaminergic neural progenitor cells (10,000 cells/well) into a 96-well U-shaped plate (Thermo Fisher Scientific, cat#174929), culturing them in a culture medium at ₃₇ °C and 5% CO2, and replacing half of the medium every 3 to 4 days after differentiation induction.

As a culture medium for dopaminergic neural spheroids of dopaminergic neural precursor cells, for example, a medium having the following composition can be used.
<Culture medium composition for dopamine neuronal spheroids>
BrainPhys Neuronal Medium (manufactured by STEMCELL Technologies, cat#ST-05793)
Dopaminergic Neuron Maturation Supplement (Thermo Fisher Scientific, cat#A3147401)
20 ng/mL BDNF (Peprotech, cat#450-02)
20 ng/mL GDNF (Peprotech, cat#450-10)
1 mM dibutyryl cAMP (Nacalai, cat#11540-74)
200 nM ascorbic acid (Nacalai, cat#03420-52)

To measure the amount of α-synuclein aggregates in neural spheroids, for example, differentiated neural spheroids are removed from the culture medium, TBS solution (Nacalai, cat. #12748-31) supplemented with 1% Trition X-100 (Nacalai, cat. #12967-32) is added, and the protein is extracted using an ultrasonic homogenizer. Protein analysis under non-reducing conditions using a Simple Western system (Protein Simple, cat. #SM-W008) with an α-synuclein antibody (Thermo Fisher Scientific, cat. #AHB0261) can be performed to quantitatively evaluate the waveform exhibited at a molecular weight of approximately 300 kD.

To measure neural vulnerability in neural spheroids, for example, differentiated dopamine neural spheroids can be transferred to a TBS solution (Nacalai, cat#12748-31) containing 1% Trition X-100 (Nacalai, cat#12967-32), proteins extracted using an ultrasonic homogenizer, and quantitatively evaluated for the waveform displayed at a molecular weight of approximately 60 kD using a Simple Western system (Protein Simple, cat#SM-W004) under reduced conditions with a tyrosine hydroxylase antibody (Millipore, cat#AB152).

To measure neuronal cell death in neural spheroids, for example, differentiated dopamine neural spheroids are transferred to a TBS solution (Nacalai, cat#12748-31) containing 1% Trition X-100 (Nacalai, cat#12967-32), proteins are extracted using an ultrasonic homogenizer, and protein analysis (Protein Simple, cat#SM-W004) using a Simple Western system under reduced conditions with a cleaved caspase 3 antibody (Cell Signaling Technology, cat#9664) can be performed to quantitatively evaluate the waveform displayed at a molecular weight of approximately 20 kD.

Abnormal neural activity in neural spheroids can be measured, for example, by measuring synchronized neural firing through imaging analysis using a fluorescent calcium probe in three-dimensionally cultured neural spheroids.
Synchronous neuronal firing in neural spheroids can be measured, for example, by imaging analysis using a measurement medium containing a fluorescent calcium probe (Molecular Devices, product name FLIPR Calcium 6 Assay Bulk Kit, cat#R8191).
As the measurement medium, for example, Hank's buffer solution (Thermo Fisher Scientific, cat#14065-056) containing 20 mM Hepes (Thermo Fisher Scientific, cat#15630-080) and 0.1% bovine serum albumin (Sigma-Aldrich, cat#A9576) can be used.

The compounds of the present invention are useful as therapeutic and/or preventive agents for central nervous system diseases associated with abnormal aggregation of proteins in the brain.
Examples of the central nervous system diseases associated with abnormal aggregates of proteins in the brain include central nervous system diseases associated with tau, α-synuclein, TDP-43, or polyglutamine.
Examples of the central nervous system diseases involving tau include Alzheimer's disease and frontotemporal lobar degeneration, while examples of diseases involving α-synuclein aggregates include Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Gaucher disease, and infantile neuroaxonal dystrophy. Examples of the central nervous system diseases involving TDP-43 include amyotrophic lateral sclerosis and frontotemporal lobar degeneration, and examples of the central nervous system diseases involving polyglutamine include Huntington's disease and spinocerebellar ataxia.
The compounds of the present application are preferably useful as therapeutic and/or preventive agents for Alzheimer's disease, frontotemporal lobar degeneration, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Gaucher's disease, infantile neuroaxonal dystrophy, amyotrophic lateral sclerosis, Huntington's disease, or spinocerebellar ataxia.
The compounds of the present application are more preferably useful as therapeutic and/or preventive agents for diseases associated with α-synuclein aggregates.
Even more preferably, the compounds of the present application are useful as therapeutic and/or preventive agents for Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Gaucher's disease, or infantile neuroaxonal dystrophy.
In the present invention, "prevention" refers to administering the active ingredient of the present invention to a healthy person who has not developed a disease, for example, with the aim of preventing the onset of a disease. "Treatment" refers to administering the compound of the present invention as an active ingredient to a person (patient) who has been diagnosed by a doctor as having developed a disease.

The compound of the present invention and a pharmaceutical containing the compound can be administered orally or parenterally, either directly or after being formulated into a suitable dosage form. Examples of dosage forms include, but are not limited to, tablets, capsules, powders, granules, liquids, suspensions, injections, patches, and poultices. The formulations are produced by known methods using pharmaceutically acceptable additives.
Depending on the purpose, the additives that can be used include excipients, disintegrants, binders, fluidizing agents, lubricants, coating agents, solubilizers, solubilizers, thickeners, dispersants, stabilizers, sweeteners, flavorings, etc. Specific examples include lactose, mannitol, crystalline cellulose, low-substituted hydroxypropyl cellulose, corn starch, partially pregelatinized starch, carmellose calcium, croscarmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, magnesium stearate, sodium stearyl fumarate, polyethylene glycol, propylene glycol, titanium oxide, talc, etc.

The most effective route of administration for treatment is desirable, and can be oral or parenteral, including intravenous, topical, inhalation, and ophthalmic administration, with oral administration being preferred. Dosage forms include tablets and injections, with tablets being preferred. The dosage and frequency of administration of these pharmaceutical compositions vary depending on the dosage form, the patient's disease and symptoms, and the patient's age and weight, and cannot be generally determined. However, typically, the amount of active ingredient per day for an adult is in the range of approximately 0.0001 to approximately 5000 mg, preferably approximately 0.001 to approximately 1000 mg, more preferably approximately 0.1 to approximately 500 mg, and particularly preferably approximately 1 to approximately 300 mg, administered once or several times a day, preferably in divided doses one to three times a day.

The compounds of the present invention and pharmaceuticals containing them can be used in combination with other drugs to enhance their effects and/or reduce their side effects. For example, they can be used in combination with drugs for treating central nervous system disorders, such as L-dopa, dopamine agonists (e.g., ropinirole hydrochloride, apomorphine hydrochloride hydrate, etc.), MAO-B inhibitors (e.g., selegiline hydrochloride, etc.), catechol-O-methyltransferase (COMT) inhibitors (e.g., entacapone, etc.), α-Syn antibodies (e.g., prasenimab, etc.), or pharmaceutically acceptable salts thereof. Hereinafter, drugs that can be used in combination with the compounds of the present invention will be abbreviated as "concomitant drugs."

The administration period for the compound of the present invention, the pharmaceutical containing the compound, and the concomitant drug is not limited, and they may be administered to the subject simultaneously or at staggered times. The compound of the present invention and the concomitant drug may also be used as a combination drug. The dose of the concomitant drug can be appropriately selected based on clinically used doses. The mixing ratio of the compound of the present invention to the concomitant drug can be appropriately selected depending on the subject, administration route, target disease, symptoms, combination, etc. For example, when the subject is a human, 0.01 to 100 parts by weight of the concomitant drug may be used per 1 part by weight of the compound of the present invention. Furthermore, to suppress side effects, the compound of the present invention may be used in combination with other drugs (concomitant drugs) such as antiemetics, hypnotics, and anticonvulsants.

The present invention will be explained in more detail below using reference examples, examples, and test examples, but the present invention is not limited to these. In this specification, the terms "example" and "reference example" may refer to compounds, such as "Example 1" meaning "compound of Example 1" and "Reference Example 1" meaning "compound of Reference Example 1." Note that the names of compounds shown in the following reference examples and examples do not necessarily conform to the IUPAC nomenclature.

To simplify the description in the specification, the following abbreviations may be used in the Reference Examples, Examples, and Test Examples.
Me: methyl, Et: ethyl, Pr: normal propyl, iPr: isopropyl, DMF: N,N-dimethylformamide, THF: tetrahydrofuran, TFA: trifluoroacetic acid, HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

The symbols used in NMR are: s for singlet, d for doublet, dd for doublet of doublets, t for triplet, td for triplet doublet, q for quartet, m for multiplet, br for broad, brs for broad singlet, brm for broad multiplet, and J for coupling constant.

The measurement conditions for the high-performance liquid chromatograph mass spectrometer (LCMS) are as follows. The observed mass spectrometry value [MS (m/z)] is indicated as MH ⁺ and the retention time is indicated as Rt (min). For each measured value, the measurement conditions used for the measurement are indicated as A or B.

Measurement condition A
Detection equipment:
MS detector:Waters ACQUITY SQ Detector
HPLC: Waters ACQUITY UPLC
Column: ACQUITY UPLC BEH C18 1.7 μm 2.1 × 30 mm
Flow rate: 0.8mL/min
Oven temperature: 40°C
Measurement wavelength: 254, 220nm
Mobile phase: Solution A 0.06% formic acid aqueous solution Solution B 0.06% formic acid acetonitrile Time program:
Step Time (min)
1 0.0-1.3 A liquid: B liquid = 98:2 to 4:96
2 1.3-1.5 A liquid: B liquid = 4:96-98; 2

Measurement condition B
Detection equipment: Agilent 1200 Series, Agilent 6110 Quadruple LCMS
Column: Xbridge C18 3.5 μm 4.6 × 50 mm
Flow rate: 1.8mL/min
Measurement wavelength: 254, 214nm
Mobile phase: Solution A: 10 mM ammonium bicarbonate aqueous solution Solution B: acetonitrile Time program:
Step Time (min)
1 0.0-1.5 A liquid: B liquid = 90:10-5:95
Oven temperature: 50°C

Reference example 1
5-(piperidin-4-yl)-2-(trifluoromethyl)pyridine
To a solution of 1-(tert-butoxycarbonyl)-1,2,3,6-tetrahydro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (6.7 g) in cyclopentyl methyl ether/water (4/1) (100 mL), 5-bromo-2-(trifluoromethyl)pyridine (4.1 g), 1,1'-bis(diphenylphosphino)ferrocene palladium dichloride (0.66 g), and cesium carbonate (12 g) were added and stirred at 100°C for 30 minutes. After cooling to room temperature, water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The resulting organic layer was washed with saturated brine and then dried over magnesium sulfate. The solvent in the dried organic layer was evaporated under reduced pressure, and the residue was then simply purified by aminosilica gel chromatography (elution solvent: hexane/ethyl acetate). The resulting crude product was dissolved in methanol (50 mL), and 10% palladium-carbon (55% wet) (0.30 g) was added. The mixture was stirred under a hydrogen atmosphere at room temperature for 3 hours. The reaction mixture was filtered through Celite and washed with methanol, and the solvent from the resulting filtrate was evaporated under reduced pressure. The residue was again dissolved in methanol (50 mL), and 10% palladium-carbon (55% wet) (0.30 g) was added. The mixture was stirred under a hydrogen atmosphere at room temperature for 3 hours. The reaction mixture was filtered through Celite and washed with methanol, and the solvent from the resulting filtrate was evaporated under reduced pressure. The resulting residue was dissolved in chloroform (10 mL), and trifluoroacetic acid (20 mL) was added. After stirring at room temperature for 1 minute, the solvent and trifluoroacetic acid were evaporated under reduced pressure. The resulting residue was purified by aminosilica gel chromatography (elution solvent: hexane/ethyl acetate → ethyl acetate/methanol) to give Reference Example 1 (3.8 g).
LC/MS ([M+H]+/Rt(min)): 231.2/0.50 (Measurement conditions A)

Reference examples 2 to 11
According to the method described in Reference Example 1, the compounds shown in Table 1 were obtained using the corresponding starting compounds.

Reference example 12
5-(azetidin-3-yl)-2-(trifluoromethyl)pyridine
Zinc powder (0.33 g) was added to a dried Schlenk tube, and the inside atmosphere was replaced with nitrogen. Under a nitrogen atmosphere, DMF (1.0 mL) and iodine (51 mg) were added and stirred at room temperature for 5 minutes. A solution of tert-butyl 3-iodoazetidine-1-carboxylate (0.19 mL) in DMF (1.0 mL) was added to the activated zinc solution, and the mixture was stirred at 40°C for 1 hour. A solution of 5-bromo-2-(trifluoromethyl)pyridine (0.23 g) and tetrakis(triphenylphosphine)palladium(0) (58 mg) in DMF (1.0 mL) was added to the prepared alkylzinc solution, and the mixture was stirred at 80°C for 2 hours. The reaction solution was cooled to 0°C, and saturated aqueous ammonium chloride solution was slowly added dropwise to quench the reaction. The resulting precipitate was removed by suction filtration and washed with ethyl acetate. The filtrate was extracted with ethyl acetate, and the resulting organic layer was washed with saturated brine and then dried over magnesium sulfate. The organic layer was evaporated under reduced pressure, and the resulting crude product was added with chloroform (1.0 mL) and trifluoroacetic acid (3.0 mL) and refluxed for 20 minutes. The mixture was cooled to room temperature, and the solvent and trifluoroacetic acid were evaporated under reduced pressure. The resulting residue was purified by aminosilica gel chromatography (elution solvent: hexane/ethyl acetate) to give Reference Example 12 (75 mg).
LC/MS ([M+H]+/Rt(min)): 203.1/0.47 (Measurement conditions A)

Reference example 13
4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-4-ol
5-Bromo-2-(trifluoromethyl)pyridine (2.0 g) was placed in a dry two-neck flask, and the interior was purged with nitrogen. Under a nitrogen atmosphere, dry THF (20 mL) was added and cooled to -78°C. A 2.6 mol/L n-butyllithium hexane solution (5.1 mL) was added dropwise, and the mixture was stirred at -78°C for 1 hour. A THF (10 mL) solution of 1-Boc-4-piperidone (1.8 g) was prepared and added dropwise to the reaction solution. After the addition was complete, the temperature was raised to room temperature. After 1 hour, the reaction solution was cooled to 0°C, and saturated aqueous ammonium chloride solution was slowly added dropwise to quench the reaction. The reaction solution was extracted with ethyl acetate, and the resulting organic layer was washed with saturated saline and then dried over magnesium sulfate. The solvent in the organic layer was evaporated under reduced pressure, and the residue was then purified by aminosilica gel chromatography (elution solvent: hexane/ethyl acetate 95:5-0:100). To the resulting crude product, chloroform (2.0 mL) and trifluoroacetic acid (4.0 mL) were added and the mixture was stirred at room temperature for 30 minutes. The solvent and trifluoroacetic acid were evaporated under reduced pressure, and the resulting residue was purified by aminosilica gel chromatography (elution solvent: hexane/ethyl acetate → ethyl acetate/methanol) to give Reference Example 13 (0.23 g).
LC/MS ([M+H]+/Rt(min)): 247.1/0.46 (Measurement conditions A)

Reference example 14
5-(4-fluoropiperidin-4-yl)-2-(trifluoromethyl)pyridine
a) Preparation of tert-butyl 4-hydroxy-4-[6-(trifluoromethyl)pyridin-3-yl]piperidine-1-carboxylate (Compound W1) 5-Bromo-2-(trifluoromethyl)pyridine (2.0 g) was placed in a dry two-neck flask, and the inside of the flask was replaced with nitrogen. Under a nitrogen atmosphere, dry THF (20 mL) was added and cooled to -78°C. A 1.6 mol/L n-butyllithium hexane solution (6.8 mL) was added dropwise, and the mixture was stirred at -78°C for 20 minutes. A THF (20 mL) solution of 1-Boc-4-piperidone (1.76 g) was prepared and added dropwise to the reaction solution. After the addition was complete, the temperature was raised to room temperature. After 1 hour, the reaction solution was cooled to 0°C, and saturated aqueous ammonium chloride solution was slowly added dropwise to quench the reaction. The reaction solution was extracted with ethyl acetate, and the resulting organic layer was washed with saturated brine and then dried over magnesium sulfate. The solvent in the organic layer was evaporated under reduced pressure, and the residue was purified by aminosilica gel chromatography (elution solvent: hexane/ethyl acetate) to obtain Compound W1 (1.18 g).
LC/MS ([M+H]+/Rt(min)): 347.2/0.93 (Measurement conditions A)

b) Preparation of 5-(4-fluoropiperidin-4-yl)-2-(trifluoromethyl)pyridine (Reference Example 14) Compound W1 (0.12 g) was dissolved in chloroform (1.5 mL), and bis(2-methoxyethyl)aminosulfur trifluoride (0.24 mL) was added, followed by stirring at room temperature for 30 minutes. The reaction solution was concentrated and simply purified by amino silica gel chromatography (elution solvent: hexane/ethyl acetate). The resulting compound was dissolved in chloroform (0.50 mL), treated with TFA (1.0 mL), and stirred at room temperature for 5 minutes. The reaction solution was concentrated, and excess TFA was removed by azeotropy with toluene. The resulting crude product was purified by amino silica gel chromatography (elution solvent: hexane/ethyl acetate → ethyl acetate/methanol) to give Reference Example 14 (18 mg).
LC/MS ([M+H]+/Rt(min)): 249.1/0.60 (Measurement conditions A)

Reference examples 15 to 21
According to the method described in Reference Example 1, the compounds shown in Table 2 were obtained using the corresponding starting compounds.

Example 1
(3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone
To a solution of Reference Example 1 (30 mg) in chloroform (1.0 mL), 3-methyloxetane-3-carboxylic acid (18 mg), triethylamine (27 μL), and HATU (60 mg) were added and stirred at room temperature for 30 minutes. Cesium carbonate (42 mg) was then added and the mixture was stirred again at room temperature for 30 minutes. The reaction solution was purified by aminosilica gel chromatography (elution solvent: hexane/ethyl acetate → ethyl acetate/methanol) to give Example 1 (41 mg).
LC/MS ([M+H]+/Rt(min)): 329.2/0.75 (Measurement conditions A)
^1H -NMR (400 MHz, DMSO-d6)δ: 8.72 (1H, d, J = 1.6 Hz), 8.02 (1H, dd, J = 2.0, 8.4 Hz), 7.84 (1H, d, J = 8.8 Hz), 4.83 (2H, dd, J = 6.4, 8.8 Hz), 4.55-4.52 (1H, m), 4.28 (2H, t, 6.8 Hz), 3.18-3.05 (2H, m), 3.01-2.93 (1H, m), 2.67 (1H, t, J = 12 Hz), 1.85-1.81 (2H, brs), 1.72-1.57 (5H, m).

The compound of Example 1 can also be synthesized by the following method.

3-methyloxetane-3-carboxylic acid (52 mg), triethylamine (0.17 mL), and a 50% ethyl acetate solution of 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide (0.40 mL) were added to a solution of the hydrochloride salt (100 mg) of Reference Example 1 in ethyl acetate (1.0 mL), and the mixture was stirred at room temperature for 1 hour. The reaction solution was purified by silica gel chromatography (elution solvent: ethyl acetate/methanol) to obtain Example 1 (93 mg).

Examples 2 to 32
The compounds shown in Table 3 were obtained using the corresponding starting compounds according to the synthesis method of Example 1.

Example 33
(3-Methylazetidin-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone
To a solution of Reference Example 1 (50 mg) in chloroform (1.0 mL), 1-(tert-butoxycarbonyl)-3-methylazetidine-3-carboxylic acid (56 mg), triethylamine (45 μL), HATU (99 mg), and cesium carbonate (71 mg) were added. After stirring at room temperature for 1 hour, the reaction solution was simply purified by amino silica gel chromatography (elution solvent: hexane/ethyl acetate → ethyl acetate/methanol) to obtain a crude product. The resulting crude product was dissolved in chloroform (1.0 mL), and TFA (1.0 mL) was added, followed by stirring at room temperature for 5 minutes. The reaction solution was concentrated and purified by amino silica gel chromatography (elution solvent: hexane/ethyl acetate → ethyl acetate/methanol) to obtain Example 33 (65 mg).
LC/MS ([M+H]+/Rt(min)): 328.2/0.60 (Measurement conditions A)
^1H -NMR (400 MHz, DMSO-d6)δ: 8.71 (1H, d, J = 2.0 Hz), 8.00 (1H, dd, J = 8.4, 2.0 Hz), 7.83 (1H, d, J = 8.4 Hz), 4.53 (1H, d, J = 12.8 Hz), 3.87 (2H, t, J = 8.4 Hz), 3.35-3.30 (1H, m), 3.16-3.07 (3H, m), 2.97 (1H, tt, J = 12, 3.6 Hz), 2.63 (1H, t, J = 12 Hz), 1.84 (2H, d, J = 12.8 Hz), 1.65-1.47 (5H, m).

The compound of Example 33 can also be synthesized by the following method.

To a solution of the hydrochloride salt (100 mg) of Reference Example 1 in ethyl acetate (1.0 mL), 1-(tert-butoxycarbonyl)-3-methylazetidine-3-carboxylic acid (97 mg), triethylamine (0.17 mL), and 50% 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide ethyl acetate solution (0.40 mL) were added and stirred at room temperature for 1 hour. The reaction solution was simply purified by silica gel chromatography (elution solvent: ethyl acetate/methanol) to obtain a crude product. The resulting crude product was dissolved in chloroform (1.0 mL), TFA (1.0 mL) was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated and purified by amino silica gel chromatography (elution solvent: ethyl acetate/methanol) to obtain Example 33 (90 mg).

Examples 34 to 40
Using the corresponding starting compounds, the compounds shown in Table 4 were obtained according to the synthesis method of Example 33. However, when the example is a salt, a salification step is included.

Example 41
(1,3-dimethylazetidin-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone hydrochloride
To a solution of Example 33 (20 mg) in THF (0.50 mL), 36% aqueous formaldehyde (19 μL) and acetic acid (5.2 μL) were added. Sodium triacetoxyborohydride (39 mg) was added while stirring at room temperature, and the mixture was stirred for 30 minutes. The reaction solution was purified by aminosilica gel chromatography (eluent: hexane/ethyl acetate → ethyl acetate/methanol). The resulting oily compound was dissolved in ethyl acetate (1 mL) and treated with a 4 mol/L solution of hydrochloric acid in ethyl acetate at room temperature to give Example 41 (15 mg).
LC/MS ([M+H]+/Rt(min)): 342.3/0.63 (Measurement conditions A)
¹ H-NMR (400 MHz, DMSO-d6)δ: 10.6 (1H, brs), 8.72 (1H, d, J = 1.6 Hz), 7.99 (1H, dd, J = 8.0, 1.6 Hz), 7.86 (1H, d, J = 8.8 Hz), 4.52 (1H, d, J = 12.8 Hz), 4.13-4.12 (2H, m), 3.92-3.90 (2H, m), 3.32-3.29 (1H, m), 3.15 (1H, t, J = 12.8 Hz), 3.00 (1H, tt, J = 12, 3.2 Hz), 2.72 (3H, s), 1.91-1.83 (2H, m), 1.68-1.50 (5H, m).

Examples 42 to 48
Using the corresponding starting compounds, the compounds shown in Table 5 were obtained according to the synthesis method of Example 41. However, when the examples are not salts, the salification step is not necessary.

Example 49
(morpholin-4-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone
To a solution of Reference Example 1 (30 mg) in chloroform (1.0 mL), morpholine-4-carbonyl chloride (18 μL) and N-ethyl-N-isopropylpropan-2-amine (34 μL) were added and stirred at room temperature for 1 hour. The reaction solution was purified by aminosilica gel chromatography (elution solvent: hexane/ethyl acetate → ethyl acetate/methanol) to give Example 49 (44 mg).
LC/MS ([M+H]+/Rt(min)): 344.2/0.79 (Measurement conditions A)
^1H -NMR (400 MHz, DMSO-d6)δ: 8.70 (1H, d, J = 2.0 Hz), 7.98 (1H, dd, J = 8.0, 2.0 Hz), 7.83 (1H, d, J = 8.0 Hz), 3.74 (2H, d, J = 13.6 Hz), 3.58 (4H, m), 3.15 (4H, m), 2.93-2.83 (3H, m), 1.82-1.79 (2H, m), 1.70-1.59 (2H, m).

Examples 50 to 59
The compounds shown in Table 6 were obtained using the corresponding starting compounds according to the synthesis method of Example 1.

Example 60
(1,4-oxazepan-4-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone
Pyridine (95 mg) was added to a solution of 1,4-oxazepane (61 mg) in dichloromethane (2.0 mL), and the mixture was cooled to 0°C. Triphosgene (72 mg) was added, and the mixture was stirred at 0°C for 30 minutes. Diisopropylethylamine (0.23 g) and Reference Example 1 (69 mg) were added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated and purified by preparative HPLC to give Example 60 (31 mg).
LC/MS ([M+H]+/Rt(min)): 357.9/1.54 (Measurement conditions B)
¹ H-NMR (500 MHz, CDCl3)δ: 8.63 (1H, s), 7.74-7.72 (1H, m), 7.66 (1H, d, J = 8.0 Hz), 3.83-3.79 (6H, m), 3.55-3.52 (4H, m), 2.95-2.90 (2H, m), 2.86-2.81 (1H, m), 2.02-2.00 (2H, m), 1.91 (2H, d, J = 11.0 Hz), 1.80-1.72 (2H, m).

Examples 61 to 63
Using the corresponding starting compounds, the compounds shown in Table 7 were obtained according to the synthesis method of Example 60.

Examples 64-65
Using the corresponding starting compounds, the compounds shown in Table 8 were obtained according to the synthesis method of Example 41. However, when the examples were not salts, the salification step was not carried out.

Example 66
[3-methyl-1-(2,2,2-trifluoroethyl)azetidin-3-yl]{4-[2-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone
To a solution of Example 34 (46 mg) in THF (3.0 mL), 2,2,2-trifluoroethyl trifluoromethanesulfonate (42 mg) and triethylamine (71 mg) were added, and the mixture was stirred at room temperature overnight. The reaction solution was concentrated and purified by preparative HPLC to obtain Example 66 (19 mg).
LC/MS ([M+H]+/Rt(min)): 410.1/1.78 (Measurement conditions B)
^1H -NMR (400 MHz, CDCl3)δ: 8.59 (1H, d, J = 4.8 Hz), 7.79 (1H, d, J = 8.0 Hz), 7.51 (1H, dd, J = 7.6, 4.8 Hz), 4.79 (1H, d, J = 13.2 Hz), 3.54-3.50 (4H, m), 3.41 (1H, d, J = 12.4 Hz), 3.26-3.17 (2H, m), 3.01 (2H, q, J = 9.2 Hz), 2.69 (1H, t, J = 12.8 Hz), 1.90 (2H, d, J = 12.8 Hz), 1.81-1.77 (1H, m), 1.69 (3H, s), 1.67-1.57 (1H, m).

Example 67
(1-cyclopropyl-3-methylazetidin-3-yl){4-[2-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone
To a solution of Example 34 (46 mg) in ethanol (4.0 mL), (1-ethoxycyclopropoxy)trimethylsilane (73 mg), acetic acid (13 mg), and sodium cyanoborohydride (44 mg) were added, followed by stirring overnight at 60° C. The reaction solution was cooled to room temperature, concentrated, and then purified by preparative HPLC to give Example 67 (22 mg).
LC/MS ([M+H]+/Rt(min)): 368.2/1.68 (Measurement conditions B)
¹ H-NMR (400 MHz, CDCl3)δ: 8.58 (1H, dd, J = 4.4, 0.8 Hz), 7.79 (1H, d, J = 8.0 Hz), 7.50 (1H, dd, J = 8.0, 4.8 Hz), 4.81 (1H, d, J = 12.8 Hz), 3.53-3.50 (3H, m), 3.38-3.31 (2H, m), 3.25-3.12 (2H, m), 2.76-2.65 (1H, m), 1.89-1.84 (3H, m), 1.71-1.61 (2H, m), 1.59 (3H, s), 0.38-0.35 (4H, m).

Test Example 1: Test to reproduce Parkinson's disease pathology (accumulation of α-synuclein aggregates) using neural spheroids in three-dimensional culture using PLA2G6 gene-mutated human iPS cells . PLA2G6 gene-mutated cells established from an iPS cell line derived from a healthy individual (clone name 201B7, obtained from the Center for iPS Cell Research and Application, Kyoto University) were cultured in StemFitAK03N medium (Ajinomoto Co., Basic03) at 37°C and 5% _CO2 .

Neural stem cells were induced from iPS cells using PSC Neuronal Induction Medium (Thermo Fisher Scientific, cat#A1647801) to create a cell stock.

The cryopreserved neural stem cells were cultured in a culture medium at 37°C in 5% CO _2. The culture medium for neural stem cells had the following composition:

Neural stem cell culture medium composition
Neurobasal medium (Thermo Fisher Scientific 2113049)
Advanced DMEM/F-12 medium (Thermo Fisher Scientific, 12634028)
Neural Induction Supplement (Thermo Fisher Scientific A1647801)

Neural stem cells (10,000 cells/well) were seeded into a 96-well U-shaped plate (Thermo Fisher Scientific, cat. #174929) and cultured in culture medium at 37°C and 5% _CO2 . Half of the culture medium was replaced every 3-4 days. The culture medium for neural spheroids had the following composition:

BrainPhys Neuronal Medium (manufactured by STEMCELL Technologies, cat#ST-05793)
NeuroCult SM1 Neuronal Supplement (manufactured by STEMCELL Technologies, cat#05711)
N2 Supplement-A (STEMCELL Technologies, cat#07152)
20 ng/mL BDNF (Peprotech, cat#450-02)
20 ng/mL GDNF (Peprotech, cat#450-10)
1 mM dibutyryl cAMP (Nacalai, cat#11540-74)
200 nM ascorbic acid (Nacalai, cat#03420-52)

The differentiated neural spheroids were removed from the culture medium and added to TBS solution (Nacalai, cat. #12748-31) containing 1% Trition X-100 (Nacalai, cat. #12967-32), and proteins were extracted using an ultrasonic homogenizer.

The amount of α-synuclein aggregates in the extracted protein was measured under non-reducing conditions using a Simple Western system with α-synuclein antibody (Thermo Fisher Scientific, cat#AHB0261) and protein analysis (Protein Simple, cat#SM-W008), and the waveform observed around a molecular weight of approximately 300 kD was quantitatively evaluated.

α-Synuclein aggregates increased rapidly from day 7 to day 9 of culture. On day 9 of culture, neural spheroids created from PLA2G6 mutant iPS cells showed more than five times the amount of α-synuclein aggregates compared to neural spheroids derived from iPS cells from healthy individuals. After day 9 of culture, the amount of aggregates showed a slower increase. The results are shown in Figure 1.

Test Example 2: Evaluation of inhibition of α-synuclein aggregate accumulation using neural spheroids created from PLA2G6 gene-mutated human iPS cells (1) Induction of differentiation of human iPS cells into neurons
Neural stem cells were induced from PLA2G6 mutant cells using PSC Neuronal Induction Medium (ThermoFisher, cat#A1647801). Neural spheroids were generated from the induced neural stem cells using a three-dimensional culture method and maintained in BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) containing NeuroCult SM1 Neuronal Supplement, N2 Supplement-A, 20 ng/mL BDNF, 20 ng/mL GDNF, 1 mM dibutyryl cAMP, and 200 nM ascorbic acid. Half of the culture medium was replaced on days 2 and 4 after differentiation induction.
The test compound was diluted with culture medium to a concentration twice that of the final concentration, and an equal volume of the double concentration solution was added to each well when half the volume was replaced 4 days after differentiation induction.

(2) Assessment of α-synuclein aggregate amount. Proteins were extracted from neural spheroids 9 days after differentiation induction using TBS solution supplemented with 1% Trition X-100, and the amount of α-synuclein aggregates was measured by protein analysis using a Simple Western system (Protein Simple, cat. #SM-W008) with an α-synuclein antibody (ThermoFisher, cat. #AHB0261).
The amount of aggregates in neural spheroids to which each test compound was added was measured, with the amount of aggregates in neural spheroids to which DMSO solution was added being set at 100%. The amount of aggregates in neural spheroids to which each test compound was added is shown in Table 9.

Test Example 3: Evaluation of reduction in α-synuclein aggregate accumulation using neural spheroids created from PLA2G6 gene-mutated human iPS cells (1) Induction of differentiation from human iPS cells to neurons
Neural stem cells were induced from PLA2G6 mutant cells using PSC Neuronal Induction Medium (ThermoFisher, cat#A1647801). Neural spheroids were generated from the induced neural stem cells using a three-dimensional culture method and maintained in BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) containing NeuroCult SM1 Neuronal Supplement, N2 Supplement-A, 20 ng/mL BDNF, 20 ng/mL GDNF, 1 mM dibutyryl cAMP, and 200 nM ascorbic acid. Half of the culture medium was replaced every 3–4 days.
The test compound was diluted with culture medium to a concentration twice that of the final concentration, and an equal volume of the double concentration solution was added to each well when half the volume was replaced 10 days after differentiation induction.

(2) Assessment of α-synuclein aggregate abundance. Proteins were extracted from neural spheroids 15 days after differentiation induction using TBS solution supplemented with 1% Trition X-100, and the amount of α-synuclein aggregates was measured by protein analysis using a Simple Western system (Protein Simple, cat. #SM-W008) with an α-synuclein antibody (ThermoFisher, cat. #AHB0261).
The aggregate amount of neural spheroids to which each test compound was added was measured. The aggregate amount of neural spheroids to which DMSO solution was added was set as 100%, and the aggregate amount data (%) upon addition of representative compounds are shown in Table 10.

Test Example 4: Test to reproduce Parkinson's disease pathology (accumulation of α-synuclein aggregates) using dopaminergic neural spheroids prepared from PLA2G6 gene-mutated human iPS cells Dopaminergic neural progenitor cells were induced from PLA2G6 gene-mutated cells using a dopaminergic neural induction kit (Thermo Fisher Scientific, cat#A3147701), and a cell stock was prepared.

Cryopreserved dopaminergic neural progenitor cells were cultured at 37°C and 5% _CO2 using a Floor Plate Cell Expansion Kit (Thermo Fisher Scientific, cat#A3165801).

First, dopaminergic neural progenitor cells (10,000 cells/well) were seeded into a 96-well U-shaped plate (Thermo Fisher Scientific cat#174929) and cultured in culture medium at 37°C and 5% _CO2 . Half of the medium was replaced every 3-4 days after differentiation induction. The culture medium for dopaminergic neural spheroids had the following composition:

BrainPhys Neuronal Medium (manufactured by STEMCELL Technologies, cat#ST-05793)
Dopaminergic Neuron Maturation Supplement (Thermo Fisher Scientific, cat#A3147401)
20 ng/mL BDNF (Peprotech, cat#450-02)
20 ng/mL GDNF (Peprotech, cat#450-10)
1 mM dibutyryl cAMP (Nacalai, cat#11540-74)
200 nM ascorbic acid (Nacalai, cat#03420-52)

The differentiated dopamine neural spheroids were removed from the culture medium and added to a TBS solution (Nacalai, cat. #12748-31) containing 1% Trition X-100 (Nacalai, cat. #12967-32), and proteins were extracted using an ultrasonic homogenizer.

The amount of α-synuclein aggregates in the extracted protein was measured using a Simple Western system with an α-synuclein antibody (Thermo Fisher Scientific, cat#AHB0261) under non-reducing conditions using protein analysis (Protein Simple, cat#SM-W008), and the waveform observed around a molecular weight of approximately 300 kD was quantitatively evaluated.

α-Synuclein aggregates increased rapidly from day 10 to day 21 of culture. On day 21 of culture, the dopaminergic neural spheroids created from PLA2G6 mutant iPS cells showed more than five times the amount of α-synuclein aggregates compared to dopaminergic neural spheroids derived from iPS cells from healthy individuals. After day 21 of culture, the amount of aggregates showed a slower increase. The results are shown in Figure 2.

Test Example 5: Evaluation of α-synuclein aggregate accumulation inhibition using dopaminergic neural spheroids generated from PLA2G6 gene-mutated human iPS cells (1) Induction of differentiation from human iPS cells to neurons. Dopaminergic neural progenitor cells were induced from PLA2G6 gene-mutated cells using a dopaminergic neural induction kit (ThermoFisher, cat#A3147701). Dopaminergic neural spheroids were generated from the induced dopaminergic neural progenitor cells using a three-dimensional culture method and maintained in BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) containing NeuroCult SM1 Neuronal Supplement, N2 Supplement-A, 20 ng/mL BDNF, 20 ng/mL GDNF, 1 mM dibutyryl cAMP, and 200 nM ascorbic acid. Half of the culture medium was replaced every 3 to 4 days.
The test compound was diluted with culture medium to a concentration twice that of the final concentration, and an equal volume of the double concentration solution was added to each well when half the volume was replaced 21 days after differentiation induction.

(2) Evaluation of α-synuclein aggregate amount Proteins were extracted from dopamine neuronal spheroids 26 days after differentiation induction using TBS solution supplemented with 1% Trition X-100, and the amount of α-synuclein aggregates was measured by protein analysis using a Simple Western system (Protein Simple, cat. #SM-W008) with an α-synuclein antibody (ThermoFisher, cat. #AHB0261).
The aggregate amount of neural spheroids to which each test compound was added was measured. The aggregate amount of neural spheroids to which DMSO solution was added was set as 100%, and the aggregate amount data (%) upon addition of representative compounds are shown in Table 11.

Test Example 6: Method for Reproducing Neurovulnerability Using Dopaminergic Neural Spheroids Prepared from PLA2G6 Gene-Mutant Human iPS Cells (1) Induction of Differentiation from Human iPS Cells to Neurons Dopaminergic neural progenitor cells were induced from PLA2G6 gene-mutant cells using a dopaminergic neural induction kit (ThermoFisher, cat#A3147701). Dopaminergic neural spheroids were prepared from the induced dopaminergic neural progenitor cells using a three-dimensional culture method and maintained in BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) containing NeuroCult SM1 Neuronal Supplement, N2 Supplement-A, 20 ng/mL BDNF, 20 ng/mL GDNF, 1 mM dibutyryl cAMP, and 200 nM ascorbic acid. Half of the culture medium was replaced every 3 to 4 days.

(2) Evaluation of tyrosine hydroxylase levels Proteins were extracted from dopamine neural spheroids 26 days after differentiation induction using TBS solution supplemented with 1% Trition X-100, and the levels of tyrosine hydroxylase were measured by protein analysis using a Simple Western system (Protein Simple, cat. #SM-W008) with a tyrosine hydroxylase antibody (Millipore, cat. #AB152). The results are shown in Figure 3.

Test Example 7: Evaluation of Improvement of Neurovulnerability Using Dopaminergic Neural Spheroids Prepared from PLA2G6 Gene-Mutant Human iPS Cells (1) Induction of Differentiation from Human iPS Cells to Neurons Dopaminergic neural progenitor cells were induced from PLA2G6 gene-mutant cells using a dopaminergic neural induction kit (ThermoFisher, cat#A3147701). Dopaminergic neural spheroids were prepared from the induced dopaminergic neural progenitor cells using a three-dimensional culture method and maintained in BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) containing NeuroCult SM1 Neuronal Supplement, N2 Supplement-A, 20 ng/mL BDNF, 20 ng/mL GDNF, 1 mM dibutyryl cAMP, and 200 nM ascorbic acid. Half of the culture medium was replaced every 3 to 4 days.
The test compound is diluted with culture medium to a concentration twice that of the final concentration, and an equal volume of the double concentration solution is added to each well when half the volume is replaced 21 days after differentiation induction.

(2) Evaluation of tyrosine hydroxylase levels Proteins were extracted from dopamine neuronal spheroids 26 days after differentiation induction using TBS solution supplemented with 1% Trition X-100, and the levels of tyrosine hydroxylase were measured by protein analysis using a Simple Western system (Protein Simple, cat. #SM-W008) with a tyrosine hydroxylase antibody (Millipore, cat. #AB152).

Test Example 8: Method for Reproducing Neuronal Cell Death Using Neural Spheroids Prepared from PLA2G6 Gene-Mutant Human iPS Cells (1) Induction of Differentiation from Human iPS Cells to Neurons Dopaminergic neural progenitor cells were induced from PLA2G6 gene-mutant cells using a dopaminergic neural induction kit (ThermoFisher, cat#A3147701). Dopaminergic neural spheroids were prepared from the induced dopaminergic neural progenitor cells using a three-dimensional culture method and maintained in BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) containing NeuroCult SM1 Neuronal Supplement, N2 Supplement-A, 20 ng/mL BDNF, 20 ng/mL GDNF, 1 mM dibutyryl cAMP, and 200 nM ascorbic acid. Half of the culture medium was replaced every 3 to 4 days.

(2) Evaluation of neuronal cell death. Starting from day 35 of differentiation induction, 10 μM dopamine was added to the culture medium. 40 days after differentiation induction, proteins were extracted from dopamine neuronal spheroids using a TBS solution containing 1% Trition X-100. Neuronal cell death was measured by protein analysis using a Simple Western system (Protein Simple, cat. #SM-W008) with a cleaved caspase 3 antibody (Cell Signaling Technology, cat. #9664). The results are shown in Figure 4.

Test Example 9: Evaluation of Neuronal Cell Death Inhibition Using Neural Spheroids Prepared from PLA2G6 Gene-Mutant Human iPS Cells (1) Induction of Differentiation from Human iPS Cells to Neurons Dopaminergic neural progenitor cells were induced from PLA2G6 gene-mutant cells using a dopaminergic neural induction kit (ThermoFisher, cat#A3147701). Dopaminergic neural spheroids were prepared from the induced dopaminergic neural progenitor cells using a three-dimensional culture method and maintained in BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) containing NeuroCult SM1 Neuronal Supplement, N2 Supplement-A, 20 ng/mL BDNF, 20 ng/mL GDNF, 1 mM dibutyryl cAMP, and 200 nM ascorbic acid. Half of the culture medium was replaced every 3 to 4 days.
Test compounds are diluted with culture medium to a concentration twice that of the final concentration, and at the time of half-exchange 35 days after differentiation induction, an equal volume of the double concentration solution is added to each well together with 10 μM dopamine.

(2) Evaluation of neuronal cell death. Proteins were extracted from dopamine neuronal spheroids 40 days after differentiation induction using TBS solution containing 1% Trition X-100. The amount of neuronal cell death was measured by protein analysis using a Simple Western system (Protein Simple, cat. #SM-W008) with cleaved caspase 3 antibody (Cell Signaling Technology, cat. #9664).

Test Example 10: Method for reproducing Parkinson's disease pathology (accumulation of α-synuclein aggregates) using neural spheroids in three-dimensional culture using GBA1 gene-mutated human iPS cells GBA1 gene homozygous mutant cells established from an iPS cell line derived from a healthy individual were cultured in StemFitAK03N medium (Ajinomoto Co., Basic03) at 37°C and 5% _CO2 .
Dopaminergic neural progenitor cells were induced from GBA1 gene homozygous mutant iPS cells using a dopaminergic neural induction kit (Thermo Fisher Scientific, cat#A3147701) to prepare a cell stock.

Cryopreserved dopaminergic neural progenitor cells were cultured at 37°C and 5% _CO2 using a Floor Plate Cell Expansion Kit (Thermo Fisher Scientific, cat#A3165801).

Dopaminergic neural progenitor cells (10,000 cells/well) were seeded into a 96-well U-shaped plate (Thermo Fisher Scientific, cat. #174929) and cultured in culture medium at 37°C and 5% _CO2 . Half of the medium was replaced every 3-4 days after differentiation induction. The culture medium for dopaminergic neural spheroids had the following composition:

BrainPhys Neuronal Medium (manufactured by STEMCELL Technologies, cat#ST-05793)
Dopaminergic Neuron Maturation Supplement (Thermo Fisher Scientific, cat#A3147401)
20 ng/mL BDNF (Peprotech, cat#450-02)
20 ng/mL GDNF (Peprotech, cat#450-10)
1 mM dibutyryl cAMP (Nacalai, cat#11540-74)
200 nM ascorbic acid (Nacalai, cat#03420-52)

The differentiated dopamine neural spheroids were removed from the culture medium and added to a TBS solution (Nacalai, cat. #12748-31) containing 1% Trition X-100 (Nacalai, cat. #12967-32), and proteins were extracted using an ultrasonic homogenizer.

The amount of α-synuclein aggregates in the extracted protein was measured under non-reducing conditions using a Simple Western system with α-synuclein antibody (Thermo Fisher Scientific, cat#AHB0261) and protein analysis (Protein Simple, cat#SM-W008), and the waveform observed around a molecular weight of approximately 300 kD was quantitatively evaluated.

α-Synuclein aggregates increased rapidly from day 21 to day 40 of culture, reaching saturation on day 40, after which no change in aggregate amount was observed. The results are shown in Figure 5.

Test Example 11: Evaluation of α-synuclein aggregate accumulation reduction using dopaminergic neural spheroids generated from GBA1 gene-mutated human iPS cells (1) Induction of differentiation from human iPS cells to neurons Dopaminergic neural progenitor cells were induced from GBA1 gene-mutated cells using a dopaminergic neural induction kit (ThermoFisher, cat#A3147701). Dopaminergic neural spheroids were generated from the induced dopaminergic neural progenitor cells using a three-dimensional culture method and maintained in BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) containing NeuroCult SM1 Neuronal Supplement, N2 Supplement-A, 20 ng/mL BDNF, 20 ng/mL GDNF, 1 mM dibutyryl cAMP, and 200 nM ascorbic acid. Half of the culture medium was replaced every 3 to 4 days.
The test compound was diluted with culture medium to a concentration twice that of the final concentration, and an equal volume of the double concentration solution was added to each well when half the volume was replaced 40 days after differentiation induction.

(2) Evaluation of α-synuclein aggregate amount Proteins were extracted from dopamine neuronal spheroids 44 days after differentiation induction using TBS solution supplemented with 1% Trition X-100, and the amount of α-synuclein aggregates was measured by protein analysis using a Simple Western system (Protein Simple, cat. #SM-W008) with an α-synuclein antibody (ThermoFisher, cat. #AHB0261).
The aggregate amount of neural spheroids to which each test compound was added was measured. The aggregate amount of neural spheroids to which DMSO solution was added was set as 100%, and the aggregate amount data (%) upon addition of representative compounds are shown in Table 12.

Test Example 12: Confirmation of abnormal synchronous firing in neural spheroids in three-dimensional culture using GBA1 gene-mutated human iPS cells GBA1 gene homozygous mutant cells established from an iPS cell line derived from a healthy individual were cultured in StemFitAK03N medium (Ajinomoto Co., Basic03) at 37°C and 5% _CO2 .

Dopamine neuron precursor cells were induced from GBA1 gene homozygous mutant iPS cells using a dopaminergic neuron induction kit (Thermo Fisher Scientific, cat#A3147701) to create a cell stock.

Cryopreserved dopaminergic neural progenitor cells are cultured at 37°C and 5% _CO2 using a Floor Plate Cell Expansion Kit (Thermo Fisher Scientific, cat#A3165801).

Dopaminergic neural progenitor cells (10,000 cells/well) are seeded into a 96-well U-shaped plate (Thermo Fisher Scientific cat#174929) and cultured in culture medium at 37°C and 5% _CO2 . Half of the medium is replaced every 3-4 days after differentiation induction. The culture medium for dopaminergic neural spheroids has the following composition:

BrainPhys Neuronal Medium (manufactured by STEMCELL Technologies, cat#ST-05793)
Dopaminergic Neuron Maturation Supplement (Thermo Fisher Scientific, cat#A3147401)
20 ng/mL BDNF (Peprotech, cat#450-02)
20 ng/mL GDNF (Peprotech, cat#450-10)
1 mM dibutyryl cAMP (Nacalai, cat#11540-74)
200 nM ascorbic acid (Nacalai, cat#03420-52)

After 40 days of differentiation induction, half of the culture medium is removed, and an equal volume of measurement medium containing a fluorescent calcium probe (Molecular Devices, product name FLIPR Calcium 6 Assay Bulk Kit, cat#R8191) is added to the remaining medium. After leaving the cells to stand for 30 minutes, measurements are performed. The measurement medium is Hank's buffer (Thermo Fisher Scientific, cat#14065-056) containing 20 mM Hepes (Thermo Fisher Scientific, cat#15630-080) and 0.1% bovine serum albumin (Sigma-Aldrich, cat#A9576). Images are captured at one frame per second.

Test Example 13: Test to evaluate the improvement of synchronous firing abnormalities using dopaminergic neural spheroids generated from GBA1 gene-mutated human iPS cells (1) Differentiation induction from human iPS cells to neurons. Dopaminergic neural progenitor cells were induced from GBA1 gene-mutated cells using a dopaminergic neural induction kit (ThermoFisher, cat#A3147701). Dopaminergic neural spheroids were generated from the induced dopaminergic neural progenitor cells using a three-dimensional culture method and maintained in BrainPhys Neuronal Medium (STEMCELL Technologies, cat#ST-05793) containing NeuroCult SM1 Neuronal Supplement, N2 Supplement-A, 20 ng/mL BDNF, 20 ng/mL GDNF, 1 mM dibutyryl cAMP, and 200 nM ascorbic acid. Half of the culture medium was replaced every 3 to 4 days.
The test compound was diluted with culture medium to a concentration twice that of the final concentration, and an equal volume of the double concentration solution was added to each well when half the volume was replaced 40 days after differentiation induction.

(2) Evaluation of synchronized firing. 44 days after differentiation induction, half of the culture medium for dopaminergic neuronal spheroids was replaced, and an equal volume of measurement medium containing a fluorescent calcium probe (Molecular Devices, product name FLIPR Calcium 6 Assay Bulk Kit, cat#R8191) was added. After 30 minutes of incubation, measurements were performed. The measurement medium used was Hank's buffer (Thermo Fisher Scientific, cat#14065-056) containing 20 mM Hepes (Thermo Fisher Scientific, cat#15630-080) and 0.1% bovine serum albumin (Sigma-Aldrich, cat#A9576). Images were captured at one frame per second.

The compounds of the present invention exhibit the effect of inhibiting or reducing the accumulation of α-synuclein aggregates, and are therefore useful as therapeutic and/or preventive agents for central nervous system disorders characterized by the effect of inhibiting or reducing the accumulation of abnormal protein aggregates in the brain. The present invention is also useful as a method for reproducing the pathology of Parkinson's disease using neural spheroids, and as a method for evaluating the amount of α-synuclein aggregates using the same.

As explained above, the compound represented by formula (1) or a pharmaceutically acceptable salt thereof exhibits the effect of inhibiting or reducing α-synuclein aggregates. Therefore, the compound represented by formula (1) or a pharmaceutically acceptable salt thereof is useful as a therapeutic and/or preventive agent for central nervous system disorders involving α-synuclein aggregates, such as Parkinson's disease and Lewy syndrome dementia.

Claims (15)

A pharmaceutical composition comprising a compound selected from the following group of compounds or a pharmaceutically acceptable salt thereof:
(3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone,
(3-methyloxetan-3-yl){4-[5-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone,
{4-[5-fluoro-6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}(3-methyloxetan-3-yl)methanone,
(3-methyloxetan-3-yl){4-[4-methyl-6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone,
(1,3-dimethylazetidin-3-yl){4-[2-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone, and (1,3-dimethylazetidin-3-yl){4-[5-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone. The pharmaceutical composition of claim 1, wherein the compound is (3-methyloxetan-3-yl){4-[6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone. The pharmaceutical composition of claim 1, wherein the compound is (3-methyloxetan-3-yl){4-[5-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone. The pharmaceutical composition of claim 1, wherein the compound is {4-[5-fluoro-6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}(3-methyloxetan-3-yl)methanone. The pharmaceutical composition of claim 1, wherein the compound is (3-methyloxetan-3-yl){4-[4-methyl-6-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone. The pharmaceutical composition of claim 1, wherein the compound is (1,3-dimethylazetidin-3-yl){4-[2-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone. The pharmaceutical composition of claim 1, wherein the compound is (1,3-dimethylazetidin-3-yl){4-[5-(trifluoromethyl)pyridin-3-yl]piperidin-1-yl}methanone. A therapeutic or preventive agent for a central nervous system disorder associated with abnormal protein aggregates in the brain, comprising the pharmaceutical composition of any one of claims 1 to 7. The therapeutic or preventive agent according to claim 8, wherein the central nervous system disease involving abnormal aggregates of proteins in the brain is a central nervous system disease involving tau, α-synuclein, TDP-43, or polyglutamine. The therapeutic or preventive agent according to claim 8, wherein the central nervous system disease involving abnormal protein aggregates in the brain is Alzheimer's disease, frontotemporal lobar degeneration, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Gaucher disease, infantile neuroaxonal dystrophy, amyotrophic lateral sclerosis, Huntington's disease, or spinocerebellar ataxia. The therapeutic or preventive agent according to claim 8, wherein the central nervous system disease involving abnormal protein aggregates in the brain is a central nervous system disease involving α-synuclein. The therapeutic or preventive agent according to claim 8, wherein the central nervous system disease involving abnormal protein aggregates in the brain is Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Gaucher disease, or infantile neuroaxonal dystrophy. The therapeutic or preventive agent according to claim 8, wherein the central nervous system disease involving abnormal protein aggregates in the brain is Parkinson's disease. The therapeutic or preventive agent according to claim 8, wherein the central nervous system disease involving abnormal protein aggregates in the brain is infantile neuroaxonal dystrophy. The therapeutic or preventive agent according to claim 8, wherein the central nervous system disease involving abnormal protein aggregates in the brain is multiple system atrophy.